bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of Calmodulin with Nuclear Structures in Starfish Oocytes and Its Role in the Resumption of Meiosis
Authors: Santella Luigia , Kyozuka Keiichiro
Published: July 2004

The resumption of meiosis in prophase‐arrested starfish oocytes is induced by the hormone 1‐methyl‐adenine, which has been shown previously to induce a calcium transient in the nucleus which at this stage is called the germinal vesicle. This transient precedes the breakdown of the germinal vesicle (GVBD). Experiments were performed to establish whether nuclear calmodulin (CaM) was involved in the progression of the meiotic cycle. CaM antagonists, antibodies, and an inhibitory peptide corresponding to the CaM‐binding domain of myosin‐light‐chain kinase have been injected into the nucleus of prophase‐arrested starfish oocytes. The antagonists failed to affect the final response to 1‐methyladenine, i.e. GVBD, although two antagonists delayed it, whereas the peptide inhibitor and the antibodies completely inhibited it. The antibodies suppressed the nuclear Ca2+ spikes that were shown by previous work to be induced by the photoreleasing of caged adenosine 3′,5′‐(cyclic)diphosphate ribose in the germinal vesicle. Immunofluorescence staining of isolated starfish oocyte nuclei with CaM antibodies showed CaM in the envelope and in the nucleolus. Immunogold labelling of oocytes revealed aggregates of CaM and of a 36‐kDa protein, of the heterogeneous ribonucleoprotein particles (hnRNP), in electron‐dense hnRNP in the nuclear matrix. 1‐Methyladenine induced the disappearance of these hnRNP from the nucleoplasm and the translocation of CaM and the 36‐kDa protein previously associated with them to the cytoplasm, prior to the breakdown of the nuclear envelope.
Myrosinase‐Binding Proteins are Derived from a Large Wound‐Inducible and Repetitive Transcript
Authors: Taipalensuu Jan , Falk Anders , Ek Bo , Rask Lars
Published: July 2004

Several non‐myrosinase proteins have been found in association with some of the myrosinases extracted from rape (Brassica napus) seed. Most of these proteins seemed to belong to a large family of proteins ranging in size over approximately 30–110 kDa, namely the myrosinase‐binding protein (MBP) family. Potentially all of these MBPs might be derived from a single large precursor, encoded by a 3.3‐kb transcript. This transcript coded for a 99‐kDa glycine‐rich protein with a highly repetitive structure. The mature 50‐kDa and 52‐kDa MBP amino‐terminal was located 255 amino acids from the putative initiation methionine. Also, a more divergently related transcript, the protein product of which was unknown, has been cloned. However, the largest open reading frame suggested a proline‐rich protein. While this transcript seemed to be expressed predominantly in seeds, the MBP transcripts were expressed in several tissues and also exhibited a responsiveness to wounding and methyl jasmonate. Both proteins exhibited significant similarities to lectins from Artocarpus integer and from Maclura pornifera.
Structural Characterization of Bovine Collectin‐43
Authors: Rothmann Annette B. , Mortensen Hanne D. , Holmskov Uffe , Højrup Peter
Published: July 2004

Bovine collectin‐43 (CL‐43), the most recently disclosed member of the collectin group, has been characterized structurally at the protein level by a combination of mass spectrometry and protein sequencing. The molecular mass of reduced CL‐43 was determined by the use of mass spectrometry to be 33.6±0.1 kDa. Furthermore, the mass spectrum showed the presence of a truncated version of the poly‐peptide, which has also previously been shown by SDS/PAGE and N‐terminal sequencing. N‐terminal Edman degradation of peptides from a tryptic digestion of native CL‐43 verified the published sequence derived from cDNA studies and partial protein sequencing [Lim, B.‐L., Willis, A. C., Reid, K. B. M., Lu, J., Lauersen, S. B., Jensenius, J. C. & Holmskov, U. (1994) J. Biol. Chem. 269, 11820–118241 with two exceptions. Using mass spectrometry and N‐terminal sequencing, a large number of post‐translational modifications were found in the collagen‐like region (repetitive Gly‐Xaa‐Yaa sequence). All proline residues located in the Yaa‐position in the collagen‐like region were found to be partially hydroxylated while all lysine residues in the Yaa position were fully hydroxylated and glycosylated. The glycosylation was determined as glycosyl‐galactosyl O‐linked to a hydroxylated lysine residue. Mass spectrometric analysis of a peptic digest of the N‐terminal tryptic peptide revealed that the three polypeptide chains were disulphide linked in a rather surprising pattern. The cysteine residues were inter‐chain disulphide linked by Cys15 in polypeptide chain 1 to Cys15 in polypeptide chain 2, Cys20 in chain 2 to Cys20 in chain 3 and Cys20 in chain 1 to Cys15 in chain 3.

The four cysteine residues at the C‐terminus were intra‐chain disulphide linked, Cys204 to Cys299 and Cys277 to Cys291, as expected for a C‐type lectin.
Partial Purification and Characterization of Nuclear Ribonuclease P from Wheat
Authors: Arends Sabine , Schön Astrid
Published: July 2004

Ribonuclease P (RNase P) from wheat nuclei has been purified over 1000‐fold, using wheat germ extract as starting material and a combination of poly(ethylenglycol) precipitation and column chromatography. The enzyme was shown to be of nuclear origin by its characteristic ionic requirements; for optimum activity it requires 0.5–1.5 mM Mg2+, which can be partly replaced by Mn2+. With about 100 kDa, wheat nuclear RNase P has the lowest molecular mass reported so far for a eukaryotic RNase P. The enzyme has an isoelectric point of 5.0 and a buoyant density of 1.34 g/ml in CsCl, suggesting the presence of a nucleic acid component; it is, however, insensitive against treatment with micrococcal nuclease. Wheat germ RNase P requires an intact tertiary structure of the pre‐tRNA substrate; its cleavage efficiency is also influenced by the presence of an intron, and by the nature of the 3′ terminus of the substrate. The apparent Km and Vmax for an intronless plant pre‐tRNATyr are 10.3 nM and 1.12 fmol/min, respectively.
13C‐NMR Studies of Transmembrane Electron Transfer to Extracellular Ferricyanide in Human Erythrocytes
Authors: Himmelreich Uwe , Kuchel Philip W.
Published: July 2004

Human erythrocytes are known to reduce ferricyanide (hexacyanoferrate) [Fe(CN)6]3‐ to ferrocyanide [Fe(CN)6]2‐ in an extracellular reaction that involves the transmembrane transfer of reducing equivalents; potentially these could be either electrons from NADH, formed in glycolysis inside the cells or transmembrane exchange of reduced solutes. The 13C‐NMR resonance of [Fe(13CN)6]3 (which was synthesised in our laboratory) was seen to be very broad while that of ferrocyanide was narrow. This phenomenon formed the basis of a simple non‐invasive procedure to study ferricyanide reduction in high‐haematocrit suspensions of erythrocytes. The method should be directly applicable to other cell types.

In a series of experiments, erythrocyte metabolism was studied in the presence of ferricyanide, using 1H, 13C, and 31P NMR spectroscopy. Incubating the cells with 13C‐labelled glucose enabled the rate of ferricyanide reduction, glucose utilisation, and lactate and bicarbonate production to be measured simultaneously. Various metabolic states were imposed as follows: glycolysis was inhibited with F− and iodoacetate; glucose transport was inhibited with phloretin and cytochalasin B; and anion transport was inhibited with dinitrostilbene 2,2′‐disulfonate and p‐chloromercuriphenyl sulfonate. Earlier work was confirmed, showing that ascorbate is intimately involved in the reduction reaction; but its main action appears not to be mediated by membrane transport but in a membrane‐associated redox‐protein complex that is functionally linked to glycolysis. Also, large differences (factors of three) in the rate of the reduction reaction were recorded in erythrocytes from different, apparently healthy, donors.
Role of the Carboxy‐Terminal Domain of Human Apolipoprotein AI in High‐Density‐Lipoprotein Metabolism
Authors: Holvoet Paul , Danloy Sophie , Collen Desiré
Published: July 2004

Cholesterol levels in high‐density lipoprotein (HDL) of transgenic mice overexpressing human apolipoprotein A1 (apoAI), a des‐(190‐243)‐apoAI deletion mutant or an apoAI‐(I‐189)‐apoAII‐(12‐77) chimera were 2.8‐fold (P<0.001), 1.3‐fold (P<0.05) and 2.2‐fold (P<0.001) higher than in control mice, respectively. Human apolipoprotein levels in apoAI and in apoAI‐(1–189)‐apoAII‐(12–77) transgenic mice were 5.2‐fold and 3.5‐fold higher than in des‐(190–243)‐apoA1 transgenic mice, whereas their HDL cholesterol levels were 2.1‐fold and 1.6‐fold higher. PAGE of HDL isolated by ultracentrifuga‐tion revealed that murine HDL migrated as 9.6‐nm and 7.2‐nm particles. Overexpression of human apoAI and apoAI‐(1‐189)‐apoAII‐(12‐77) resulted in the production of polydisperse HDL (9.6, 9.2, 8.4 and 7.2 nm) particles, whereas overexpression of des‐(190–243)‐apoAI primarily resulted in an increase of 7.2‐nm particles. The fractional catabolic rates of human apoAI and apoAI‐(1‐189)‐apoAII‐(12‐77) were very similar, whereas that of des‐(190‐243)‐apoAI was 4.9‐fold higher. The endogenous production rates of human apoAI, des‐(190‐243)‐apoAI and apoAI‐(1‐189)‐apoAII‐(12‐77) in transgenic mice were very similar. It is concluded that deletion of the carboxy‐terminal domain of apoAI reduces its lipoprotein association, resulting in the production of small, phospholipid‐rich HDL particles that are cleared more rapidly. Substitution of the carboxy‐terminal helices of apoAI with helices of apoAII restores lipoprotein association, resulting in the production of HDL, which migrates as human HDL3 and HDL2. Although the carboxy‐terminal domain of the chimera contained more than 80% of the amino acid sequence of apoAII, its HDL‐distribution profile in transgenic mice was very similar to that of human apoAI. This study demonstrates the importance of the helical structure of apoAI of the carboxy‐terminal domain of apoAI, rather than of its exclusive amino acid sequence, in HDL metabolism.
Membrane‐Type‐2 Matrix Metalloproteinase Can Initiate the Processing of Progelatinase A and is Regulated by the Tissue Inhibitors of Metalloproteinases
Authors: Butler Georgina S. , Will Horst , Atkinson Susan J. , Murphy Gillian
Published: July 2004

Membrane‐type‐1 matrix metalloproteinase has been identified as an activator of the matrix metalloproteinase progelatinase A at cell surfaces. We report here that a soluble active form of membrane‐type‐2 matrix metalloproteinase can also process progelatinase A in a comparable fashion to the type‐1 at rates which are dependent on the concentration of the proenzyme. Activation is inhibited by tissue inhibitors of metalloproteinases TIMP‐2 and TIMP‐3, but only partially by TIMP‐1. These results suggest that cellular activation of progelatinase A may be initiated by different members of the membrane‐type matrix metalloproteinase family depending on tissue distribution.
Spectroscopic Probes of the Individual and Combined Effects of Triton X‐100 and Chloroform on Serum Albumins and Serum‐Albumin · Bilirubin Complexes
Authors: Patra Samir Kumar , Pal Medini Kanta
Published: July 2004

The effects of the non‐ionic surfactant Triton X‐100 on the biphasic induced CD spectra of bilirubin complexes of human and bovine serum albumins (HSA and BSA) are divergent. While Triton X‐100 inverts the induced CD spectrum of HSA · bilirubin, this surfactant enhances the ellipticity values of induced CD of BSA · bilirubin without inversion. The effect of Triton X‐100 on the characteristic ultraviolet‐CD spectra of the albumins are similar; both the albumins are denatured from their native globular structures. The anionic surfactant SDS, unlike non‐ionic Triton X‐100, dislodges the ligand from its protein complexes, indicating that both electrostatic and hydrophobic forces are involved in binding of bilirubin to the albumins.

The aprotic solvent chloroform inverts the biphasic induced CD spectra of HSA · bilirubin and BSA · bilirubin, whereas CHCl3 has relatively little effect on the ultraviolet CD spectra of the albumins. The combined effect of Triton X‐100 and CHCl3 shows that the effect of CHCl3 predominates over that of Triton X‐100. The perturbing effects of Triton X‐100 and CHCl3 on the CD or induced CD spectra of the proteins or their bilirubin complexes are reversible, and independent of the order in which components were added. The observations suggest that the denaturation of the albumins by Triton X‐100 or solvation of CHCl3 within albumins markedly alter the internal topography or dynamics of the receptor sites, triggering alterations of the chirality of the bound pigment in sign and/or magnitude.
Characterization of a Phosphoprotein whose mRNA is Regulated by the Mitogenic Pathways in Dog Thyroid Cells
Authors: Wilkin Fraçoise , Suarez‐Huerta Nathalie , Robaye Bernard , Peetermans Julien , Libert Frederick , Dumont Jacques E. , Maenhaut Carine
Published: July 2004

We have isolated cDNA clones encoding the dog and human forms of a novel protein whose function is still unknown. Sequence analysis indicates that dog clone c5fw protein contains 343 amino acid residues, several potential phosphorylation sites, and two of the 12 conserved subdomains (VIII and IX) that fold into a common catalytic core structure of the large family of protein kinases. Human clone c5fw shares 95% amino acid identity with its dog counterpart. We have also isolated another human‐related clone c5fw sharing 70% amino acid identity with the dog sequence. We transiently expressed c‐myc epitope‐tagged clone c5fw protein in COS‐7 cells and infected thyrocytes in primary culture with a recombinant adenovirus containing clone c5fw cDNA (adenovirus c5fw).In both experiments, a 46‐kDa protein was detected and subsequently more extensively characterized. By two‐dimensional gel electrophoresis and V8 protease digestion, we showed that this overexpressed protein is phosphorylated on different sites. Moreover, cells stimulated with thyrotropin or epiderinal growth factor, thyrotropin and fetal calf serum increased the level of clone c5fw protein produced after infection by adenovirus containing clone c5fw. The disappearance of this 46‐kDa protein after 1 h of puromycin treatment indicates that it is a labile protein. Immunofluorescence and subcellular fractionation analysis have revealed that c‐myc‐tagged clone c5fw was insoluble and localized mainly in the cytoplasm, in the form of granules.
Characterisation of the Rat Tissue‐Type Plasminogen Activator Gene Promoter
Authors: Leonardsson Göran , Ny Tor
Published: July 2004

Tissue‐type plasminogen activator (tPA) activates plasminogen to the active protease plasmin and is implicated in many biological processes that require extracellular proteolysis. In rat ovarian cells, gonadotropins induce the tPA gene by a CAMP‐dependent pathway and this induction correlates with the time of follicular rupture. We have previously identified several promoter elements within the first 621 bp of the rat tPA promoter that are important for constitutive and CAMP‐induced expression of the gene, including a cAMP responsive element (CRE), a nuclear factor 1 (NF1) element, a SPI‐binding site and a G+C‐rich box. In this report we have extended our study by analysing promoter constructs, ranging in size from 7.7 kb to 135 bp fused to the luciferase reporter gene. Transient transfection analysis of rat granulose cells and human 293 cells, reveal that the proximal 268 bp of the promoter is enough to confer high basal and CAMP‐induced expression of the gene. At position‐162 to ‐172, between the previously identified CRE and NF1 sites, a novel TAAT‐containing promoter element was identified. Mutational inactivation of the TAAT motif indicates that this element is important for both constitutive and CAMP‐induced expression of the gene, and for the binding of a presumably novel nuclear factor that we have termed tPA promoter factor‐1 (tPF‐1).
Man9‐Mannosidase from Pig Liver is a Type‐II Membrane Protein That Resides in the Endoplasmic Reticulum
Authors: Bieberich Erhard , Treml Kornelia , Völker Christof , Rolfs Andreas , Kalz‐Füller Burga , Bause Ernst
Published: July 2004

Man9‐mannosidase, one of three different α1,2‐exo‐mannosidases known to be involved in N‐linked oligosaccharide processing, has been cloned in λgt10, using a mixed‐primed pig liver cDNA library. Three clones were isolated which allowed the reconstruction of a 2731‐bp full‐length cDNA. The cDNA construct contained a single open reading frame of 1977 bp, encoding a 659‐residue polypeptide with a molecular mass of ≈73 kDa. The Man9‐mannosidase specificity of the cDNA construct was verified by the observation that all peptide sequences derived from a previously purified, catalytically active 49‐kDa fragment were found within the coding region. The N‐terminus of the 49‐kDa fragment aligns with amino acid 175 of the translated cDNA, indicating that the catalytic activity is associated with the C‐terminus. Transfection of COS 1 cells with the Man9‐mannosidase cDNA gave rise to a > 30‐fold over‐expression of a 73‐kDa protein whose catalytic properties, including substrate specificity, susceptibility towards α‐mannosidase inhibitors and metal ion requirements, were similar to those of the 49‐kDa enzyme fragment. Thus deletion of 174 N‐terminal amino acids in the 73‐kDa protein appears to have only marginal influence on the catalytic properties.

Structural and hydrophobicity analysis of the coding region, as well as the results from tryptic degradation studies, point to pig liver Man9‐mannosidase being a non‐glycosylated type‐II transmembrane protein. This protein contains a 48‐residue cytosolic tail followed by a 22‐residue membrane anchor (which probably functions as internal and non‐cleavable signal sequence), a lumenal ≈100‐residue‐stem region and a large 49‐kDa C‐terminal catalytic domain. As shown by immuno‐fluorescence microscopy, the pig liver enzyme expressed in COS 1 cells, is resident in the endoplasmic reticulum, in contrast to COS 1 Man9‐mannosidase from human kidney which is Golgi‐located [Bieberich, E. & Bause, E. (1995)Eur. J. Biochem. 233, 644–649]. Localization of the porcine enzyme in the endoplasmic reticulum is consistent with immunoelectron‐microscopic studies using pig hepatocytes. The different intracellular distribution of pig liver and human kidney Man9‐mannosidase is, therefore, enzyme‐specific rather than a COS‐1‐cell‐typical phenomenon. Since we oberserve ≈81% sequence similarity between the two α‐mannosidases, we deduce that the localization in either endoplasmic reticulum or Golgi is likely to be sequence‐dependent.
The in Vitro Phosphorylation of P53 by Calcium‐Dependent Protein Kinase C
Authors: Delphi Christian , Huang Kuo‐Ping , Scotto Christian , Chapel Agnés , Vincon Mathilde , Chambaz Edmond , Garin Jérôme , Baudier Jacques
Published: July 2004

We show that, in vitro, Ca2+‐dependent protein kinase C (PKC) phosphorylates recombinant murine p53 protein on several residues contained within a conserved basic region of 25 amino acids, located in the C‐terminal part of the protein. Accordingly, synthetic p53‐(357–381)‐peptide is phosphorylated by PKC at multiple Ser and Thr residues, including Ser360, Thr365, Ser370 and Thr377. We also establish that p53‐(357–381)‐peptide at micromolar concentrations has the ability to stimulate sequence‐specific DNA binding by p53. That stimulation is lost upon phosphorylation by PKC. To further characterise the mechanisms that regulate PKC‐dependent phosphorylation of p53‐(357–381)‐peptide, the phosphorylation of recombinant p53 and p53‐(357–381)‐peptide by PKC were compared. The results suggest that phosphorylation of full‐length p53 on the C‐terminal PKC sites is highly dependent on the accessibility of the phosphorylation sites and that a domain on p53 distinct from p53‐(357–381)‐peptide is involved in binding PKC. Accordingly, we have identified a conserved 27‐amino‐acid peptide, p53‐(320–346)‐peptide, within the C‐terminal region of p53 and adjacent to residues 357‐381 that interacts with PKC in vitro. The interaction between p53‐(320–346)‐peptide and PKC inhibits PKC autophosphorylation and the phosphorylation of substrates, including p53‐(357–381)‐peptide, neurogranin and histone H1. Conventional Ca2+‐dependent PKC α, β and γ and the catalytic fragment of PKC (PKM) were nearly equally susceptible to inhibition by p53‐(320–346)‐peptide. The Ca2+‐independent PKC  was much less sensitive to inhibition. The significance of these findings for understanding the in vivo phosphorylation of p53 by PKC are discussed.
The Product of the Molybdenum Cofactor Gene mobB of Escherichia Coli is a GTP‐Binding Protein
Authors: Eaves Deborah J. , Palmer Tracy , Boxer David H.
Published: July 2004

The mob mutants of Escherichia coli are pleiotropically defective in molybdoenzyme activities because they are unable to catalyse the conversion of molybdopterin to molybdopterin guanine dinucleotide, the active form of the molybdenum cofactor. The mob locus comprises two genes. The product of mobA, protein FA, has previously been purified to homogeneity and is able to restore molybdoenzyme activities following incubation with cell extracts of mob strains. The mobB gene, although not essential for the biosynthesis of active molybdoenzymes, encodes a protein which, sequence analysis strongly suggests, contains a nucleotide‐binding site. We have overproduced the products of both the mobA and mobB genes in engineered E. coli strains and purified each to homogeneity. The preparation of protein FA (MobA) is simpler than that previously published and produces a much greater yield of active protein. The isolated MobB protein, which is dimeric in solution, acts in the presence of protein FA, to enhance the level of nitrate reductase activation achieved on incubation with mob cell extracts. Equilibrium dialysis experiments show that purified MobB binds 0.83 mol GTP/mol protein with a Kd of 2.0 μM. Isolated MobB also catalyses a low GTPase activity (turnover number of 3X10−3 min−1) with a Km for GTP to GDP of 7.5 μM. Under the conditions tested, protein FA did not affect the GTP‐binding or GTPase activity of MobB. Intrinsic (tryptophan) protein fluorescence measurements show that MobB also binds the nucleotides ATP, TTP and GDP, but with lower affinity than GTP. These results are consistent with a model whereby MobB binds the guanine nucleotide which is attached to molybdopterin during the biosynthesis of the molybdenum cofactor.
The Glycosylation of the Aspartic Proteinases from Barley (Hordeum Vulgare L.) and Cardoon (Cynara Cardunculus L.)
Authors: Costa Júlia , Ashford David A. , Nimtz Manfred , Bento Isabel , FrazÃo Carlos , Esteves Cristina L. , Faro Carlos J. , Kervinen Jukka , Pires Euclides , VerÍssimo Paula , Wlodawer Alexander , Carrondo Maria Arménia
Published: July 2004

Plant aspartic proteinases characterised at the molecular level contain one or more consensus N‐glycosylation sites [Runeberg‐Roos, P., Törmäkangas, K. & Östman, A. (1991) Eur. J. Biochem. 202, 1021–1027; Asakura, T., Watanabe, H., Abe, K. & Arai, S. (1995) Eur. J. Biochem. 232, 77–83; Veríssimo, P., Faro, C., Moir, A. J. G., Lin, Y., Tang, J. & Pires, E. (1996) Eur. J. Biochem. 235, 762–7681. We found that the glycosylation sites are occupied for the barley (Hordeum vulgare L.) aspartic proteinase (Asn333) and the cardoon (Cynara cardunculus L.) aspartic proteinase, cardosin A (Asn70 and Asn363). The oligosaccharides from each site were released from peptide pools by enzymatic hydrolysis with peptide‐N‐glycanase A or by hydrazinolysis and their structures were determined by exoglycosidase sequencing combined with matrix‐assisted laser desorption/ionization time of flight mass spectrometry. It was observed that 6% of the oligosaccharides from the first glycosylation site of cardosin A are of the oligomannose type. Modified type glycans with proximal Fuc and without Xyl account for about 82%, 14% and 3% of the total oligosaccharides from the first and the second glycosylation sites of cardosin A and from H. vulgare aspartic proteinase, respectively. Oligosaccharides with Xyl but without proximal Fuc were only detected in the latter proteinase (4%). Glycans with proximal Fuc and Xyl account for 6%, 86% and 92% of the total oligosaccharides from the first and second glycosylation sites of cardosin A and from H. vulgure aspartic proteinase, respectively.
Screening Mixtures for Biological Activity by NMR
Authors: Meyer Bernd , Weimar Thomas , Peters Thomas
Published: July 2004

Development of new drugs often involves the screening of compound libraries for biological activity. Currently, the biologically active component can only be identified if either a pure compound is being tested or if the components of a mixture are spatially separated, for example, on beads. Here, we present an NMR technique based on the transferred nuclear Overhauser effect (transfer NOE) that allows identification and structural characterization of biologically active molecules from a mixture. As an example we demonstrate that from mixtures of oligosaccharides only α‐l‐Fuc‐(1→6)‐β‐d‐GlcNAc‐OMe binds to Aleuria aurantia agglutinin. The sign of transferred NOEs is opposite to NOEs of small molecules that do not bind to the protein and, thus, an unequivocal identification of molecules with binding activity is possible. Normally, the selection of bound ligands is further facilitated in that the absolute intensity of transfer NOEs is much greater than that of NOEs of non‐binding molecules. In addition, transfer NOEs provide information on the three‐dimensional structure of the ligands in the bound state. Therefore, measuring transfer NOEs of mixtures of small molecules in the presence of large molecules, like proteins, should significantly enhance the options for screening mixtures of compounds for biological activity.
Cloning of the Mouse Laminin α4 cDNA. Expression in a Subset of Endothelium
Authors: Frieser Michael , Nöckel Holger , Pausch Friederike , Röder Christine , Hahn Astrid , Deutzmann Rainer , Sorokin Lydia M.
Published: July 2004

Endothelial cells express a 400‐kDa or 240‐kDa laminin α chain, depending on their tissue of origin or physiological state [1, 2]. Using differential display and subsequent screening of a mouse endothelial cell cDNA library we here identify the gene coding for the 240‐kDa laminin chain as the laminin α4 gene. The complete mouse laminin α4 cDNA sequence is reported and compared with other laminin α chains. In situ hybridization of embryonic and new born mouse tissues revealed expression of laminin α4 mRNA in a subset of endothelium, in particular aortic endothelium, endocardium and endothelium of blood vessels in the skin and in the brain. Strong laminin α4 expression by aortic endothelia was confirmed by data obtained from cultured bovine aortic endothelial cells (BAEC). Isolation of laminin from BAEC conditioned medium revealed a Y‐shaped molecule in rotary shadowing. Subsequent sequencing of BAEC laminin resulted in laminin α4, β1 and γl amino acid sequences, confirming that laminin α4 is one of the major laminin α chains expressed by aortic endothelium not only in the mouse. In addition, strong laminin α4 mRNA expression occurred in peripheral nerves, cardiac muscle, fat, the dermis of the skin and lung stroma of mouse tissues. The data demonstrate a cytokine and progesterone‐regulated differential expression of laminin α4 mRNA in mouse endothelium, suggesting a distinct functional role for this laminin chain in endothelium.
Transcription Analysis of the Genes tcdA‐E of the Pathogenicity Locus of Clostridium Difficile
Authors: Hundsberger Thomas , Braun Veil , Weidmann Manfred , Leukel Petra , Sauerborn Markus , Von Eichel‐Streiber Christoph
Published: July 2004

To analyse the transcription pattern of the five tcdA‐E genes of the pathogenicity locus (PaLoc) of Clostridium difficile a protocol was established to purify RNA from strain VPI10463. Transcription analysis of the five tcdA‐E genes showed that they were all transcribed. In the early exponential phase, a high level of tcdC and low levels of tcdA,B,D,E transcripts were detectable; this was inverted in the stationary phase, suggesting that TcdC might have a negative influence on transcription of the other genes. Three transcription initiation sites, one for tcdA and two for tcdB were determined by primer extension analysis. Readthrough transcripts from outside the locus were not obtainable, so that parts of the transcription of tcdD, tcdB, tcdA and tcdC must occur by monocistronic transcription. Within the locus all possible intergenic readthrough transcripts were detectable except that between tcdC and tcdA, a stretch of DNA interrupted by a functional transcription terminator. Thus we found mono‐ and polycistronic transcription of tcdA and tcdB to occur which should lead to production of a surplus of tcdA over tcdB transcripts. This would explain the surplus of TcdA over TcdB expression observed in vitro. Due to its basic nature and similarity to BcnA of Clostridium perfringens and to Orf‐22 of Clostridium botulinum, TcdD is most probably a regulatory protein with DNA‐binding properties. On the basis of the presented study we discuss a model for the growth‐phase‐related, coordinate regulation of toxin expression wherein tcdC has a negative and tcdD a positive regulatory function on transcription of the tcdD,B,E and tcdA genes.
Cloning and Characterization of Manduca Sexta and Plutella Xylostella Midgut Aminopeptidase N Enzymes Related to Bacillus Thuringiensis Toxin‐Binding Proteins
Authors: Denolf Peter , Hendrickx Koen , Van Damme José , Jansens Stefan , Peferoen Marnix , Degheele Danny , Van Rie Jeroen
Published: July 2004

We report the purification, cloning and characterization of an aminopeptidase N from the midgut epithelium of Manduca sexta that binds Cry1Ab5, an insecticidal crystal protein [ICP] from Bacillus thuringiensis. Sequence information derived from this M. sexta aminopeptidase N was used for the cloning of an aminopeptidase N from the midgut brush‐border membrane of Plutella xylostella, an insect species of which some populations acquired resistance against Cry1Ab5. Affinity chromatography on a Cry1Ab5 matrix was used to isolate a 120‐kDa glycoprotein from the larval midgut of the lepidopteran M. sexta. On ligand blots the purified 120‐kDa protein discriminates between the lepidopteran‐specific Cry1Ab5 and the coleopteran‐specific Cry3A δ‐endotoxin. Internal amino acid sequences from the 120‐kDa protein were used for the design of degenerate oligonucleotides. From a nested PCR with M. sexta midgut cDNA as template, a DNA fragment was obtained which shows similarity to prokaryotic and eukaryotic aminopeptidase N genes. This PCR fragment was used to screen cDNA libraries of larval midguts from M. sexta and P. xylostella. From the M. sexta midgut cDNA library a 2973‐bp nucleotide sequence was cloned. The ORF of the sequence encodes a 942‐residue aminopeptidase N (M. sexta Apn2) containing two hydrophobic regions. The NH2‐terminal hydrophobic region corresponds to a secretory signal sequence and the COOH‐terminal hydrophobic region is typical of glycosylphosphatidylinositol (glycosyl‐PtdIns)‐anchored proteins. Low‐stringency hybridization of the P. xylostella midgut cDNA library with M. sexta apn2 probes enabled the isolation of a 3118‐bp sequence with an ORF encoding a 946‐residue preproprotein. This aminopeptidase N (P. xylostella Apn1) displays 61 % amino acid identity to M. sexta Apn2 and contains a COOH‐terminal signal peptide for glycosyl‐PtdIns anchor addition. Both M. sexta Apn2 and P. xylostella Apn1 contain four Cys residues, which are highly conserved among eukaryotic aminopeptidase N molecules. Treatment of Sf9 cells expressing the P xylostella apn1 gene with PtdIns‐specific phospholipase C demonstrated that P. xylostella Apn1 is attached to the insect cell membrane by a glycosyl‐PtdIns anchor.
Structure and Expression of the Gene Encoding Secretor‐Type Galactoside 2‐α‐l‐fucosyltransferase (FUT2)
Authors: Koda Yoshiro , Soejima Mikiko , Wang Baojie , Kimura Hiroshi
Published: July 2004

The expression and secretion of ABO antigens in epithelial cells of glands are controlled by secretor‐type α(1,2) fucosyltransferase activity. We have examined the expression of the secretor‐type α(1,2)fucosyltransferase gene (FUT2) and a pseudogene of FUT2 (Sec1) in several tumor cell lines by northern blot and/or reverse‐transcription‐PCR (RT‐PCR) analyses. Transcripts of FUT2 were found in total RNA from ovarian, gastric and colonic cancer cell lines but not from six leukemic cell lines, including erythroleukemic HEL cells, by RT‐PCR. On the other hand, RT‐PCR indicated that Sec1 was expressed in all these tumor cells, including all hematopoietic cells studied. Northern blot analysis indicated that FUT2 transcripts with a similar size (3.3 kb) were expressed in cancer cell lines. Rapid amplification of cDNA ends suggested that the entire FUT2 cDNA is 3.1‐kb long and has two Alu repetitive elements in its 3′ untranslated region, including an inverted repeat. The mRNA, therefore, may form a large stem‐and‐loop structure (1.2 kb). Each stem contains about 300 bases, the loop contains 640 bases, and the percentage of complementary nucleotide sequences in the stem region is 85%. The presence of a large stem‐and‐loop structure in the 3′ untranslated region may regulate the level of the FUT2 transcript by effecting the stability of the mRNA.
Identification of an Adenine–Nucleotide–Binding Site on Interferon α2
Authors: Olcott Michael C. , Haley Boyd E.
Published: July 2004

Using 32P‐labeled 2‐azidoadenosine 5′‐triphosphate (2N3ATP) and 8‐azidoadenosine 5′‐triphosphate (8N3ATP), we have identified a site on human interferon α2 (IFN‐α2) that binds adenine nucleotides. The results from saturation and competition experiments demonstrated the specificity of the nucleotide interaction. Half‐maximal saturation of IFN‐α2 was observed at 10 μM 2N3ATP or 35 μM 8N3ATP. ATP effectively decreased photoinsertion of both photoaffinity analogs of ATP. Photoinsertion of 8N3ATP was enhanced by MgCl2, independent of the ionic strength, and exhibited an optimum pH between 7.0 and 7.5. Immobilized‐Al3+ affinity chromatography and HPLC were used to purify the modified peptides from IFN‐α2 that had been photolabeled with 8N3ATP and digested with trypsin or chymotrypsin. Overlapping‐sequence analysis localized the sites of photoinsertion to the region corresponding to Lys121‐Tyr135 in the amino acid sequence of IFN‐α2, which almost perfectly overlaps a nuclear‐localization signal (R120KYFQRITLYLKEKKY135).
The Prothoracicotropic Hormone Bombyxin has Specific Receptors on Insect Ovarian Cells
Authors: Fullbright George , Lacy Eric R. , Büllesbach Erika E.
Published: July 2004

Bombyxin II, a product of the brain of the adult silkmoth, Bombyx mori, binds to ovarian cells of three different species of lepidoptera, i.e. B. mori (silkmoth), Samia Cynthia ricini (ailanthus moth), and an ovarian cell line of Spodoptera frugiperda (Sf9) (fall armyworm). Crude Sf9 cell membrane preparations were used to show that the purported bombyxin receptor binds its ligand in a specific, saturable, and reversible manner. The dissociation constant of the bombyxin‐receptor complex is 260 ± 90 pM. Quantitative binding studies and Scatchard analysis suggest that every Sf9 cell displays 20000 receptors on the surface. The cross‐linked bombyxin‐receptor ligand complex has an apparent molecular mass of about 300 kDa as determined by SDSIPAGE. Reduction causes the bombyxin receptor to dissociate into two subunits with molecular masses of 90 kDa and 116 kDa. The size and subunit structure of the putative bombyxin receptor on Sf9 cells show some similarities to the mammalian insulin receptor.
Type‐1 Plasminogen‐Activator Inhibitor
Authors: Egelund Rikke , Schousboe Susanne L. , Sottrup‐Jensen Lars , Rodenburg Kees W. , Andreasen Peter A.
Published: July 2004

We have analysed the susceptibility of latent, active, reactive‐centre‐cleaved and plasminogen‐activator‐complexed type‐1 plasminogen‐activator inhibitor (PAI‐1) to the non‐target proteinases trypsin, endoproteinase Asp‐N, proteinase K and subtilisin. This analysis has allowed us to detect conformational differences between the different forms of PAI‐1 outside the reactive‐centre loop and β‐sheet A. Proteinase‐hypersensitive sites were clustered in three regions. Firstly, susceptibility was observed in the region around α‐helix E, β‐strand 1A, and the flanking loops, which are believed to form tlexible joints during movements of β‐sheet A. Secondly, hypersensitive sites were observed in the loop between α‐helix 1 and β‐strand 5A. Thirdly, the gate region, encompassing β‐strands 3C and 4C, was highly susceptible to trypsin in latent PAI‐1, but not in the other conformations. The digestion patterns differed among all four forms of PAT‐1, indicating that each represents a unique conformation. The differential proteolytic susceptibility of the flexible‐joint region may be coupled to the differential affinity to vitronectin, binding in the same region. The analysis also allowed detection of conformational differences between reactive‐centre‐cleaved forms produced under different solvent conditions. The digestion pattern of plasminogen‐activator‐complexed PAI‐1 was different from that of active PAI‐1, but indistinguishable from that of one of the reactive‐centre‐cleaved forms, as the complexed and this particular cleaved PAI‐1 were completely resistant to all the non‐target proteinases tested. This observation is in agreement with the notion that complex formation involves reactive‐centre cleavage and a large degree of insertion of the reactive‐centre loop into β‐sheet A. Our analysis has allowed the identification of some flexible regions that appear to be implicated in the conformational changes during the movements of β‐sheet A and during the inhibitory reaction of serpins with their target proteinases.
Solution Structure of Neuropeptide Tyrosine 13–36, a Y2 Receptor Agonist, as Determined by NMR
Authors: Labelle Martin , St‐Pierre Serge , Savard Roland , Boulanger Yvan
Published: July 2004

The three‐dimensional structure of neuropeptide tyrosine (NPY) 13–36, a specific Y2 receptor agonist, has been investigated by two‐dimensional 1H‐NMR spectroscopy in solution. Analysis of the double‐quantum‐filtered correlation spectroscopy (DQFCOSY), total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY) spectra provided a complete assignment of the proton signals. The interproton connectivities observed in the NOESY spectra comprised 166 intraresidue and 95 interresidue distance ranges which were used as constraints for molecular modeling by distance geometry, simulated annealing and energy minimization. The optimal structures are characterized by a helical C‐terminal fragment Leu30–Tyr36 and a wide loop from Leu17 to Ser22. The structure of NPY 13–36 is analogous to the structure of NPY under the same solvent conditions. Comparison with other reported Y2 agonists suggests that the helical Leu30–Tyr36 fragment is the most critical for activity.
Tissue‐Specific and Stage‐Specific Expression of Two Silkworm Ecdysone Receptor Isoforms
Authors: Kamimura Manabu , Tomita Shuichiro , Kiuchi Makoto , Fujiwara Haruhiko
Published: July 2004

Previously, we isolated a cDNA clone for the ecdysone receptor B1 isoform of the silkworm, Bombyx mori (BmEcR‐B1). Here we report the cloning of a cDNA that encodes the Bombyx ecdysone receptor A isoform (BmEcR‐A) and mRNA expression of the two BmEcR isoforms during molting and metamorphosis. At larval‐pupal transformation, mRNA expression of BmEcR‐B1 was predominant in most tissues examined, including three larval tissues (midgut, epidermis, and fat body) and the wing imaginal disc. The anterior silk gland was the only tissue where BmEcR‐A was predominant. These expression patterns were different from observations demonstrated in Drosophila. In the anterior silk gland, both EcR isoforms were expressed synchronously during the fifth larval instar, while expression of the A isoform preceded that of the B1 isoform by two days in the fourth instar. Precedence of BmEcR‐A during the fourth instar and synchronization of both isoforms during the fifth instar were also observed in the middle and posterior silk glands, suggesting that transcription of BmEcR in the silk gland is regulated differently in these two instars. In the cultured anterior silk glands of day 0 of the fifth instar, transcription of BmEcR‐A and BmEcR‐B1 was induced dose dependently by more than 5 ng/ml 20‐hydroxyecdysone. BmEcR‐A and BmEcR‐B1 mRNAs were induced within 2 h and 1 h, respectively, of the addition of 20‐hydroxyecdysone. These results suggest that the increase of BmEcR mRNAs during the fifth instar is induced in vivo by a small increase in ecdysteroids.
Comparative Biochemical Characterization of the Iron‐Only Nitrogenase and the Molybdenum Nitrogenase from Rhodobacter Capsulatus
Authors: Schneider Klaus , Gollan Ute , Dröttboom Melanie , Selsemeier‐Voigt Sabine , Müller Achim
Published: July 2004

The component proteins of the iron‐only nitrogenase were isolated from Rhodobacter capsulatus (ΔnifHDK, μModABCD strain) and purified in a one‐day procedure that included only one column‐chromatography step (DEAE‐Sephacel). This procedure yielded component 1 (FeFe protein, Rc1Fe), which was more than 95% pure, and an approximately 80 % pure component 2 (Fe protein, Rc2Fe). The highest specific activities, which were achieved at an Rc2Fe/Rc1Fe molar ratio of 40:1, were 260 (C2H4 from C2H2), 350 (NH3 formation), and 2400 (H2 evolution) nmol product formed min−1 mg protein−1. The purified FeFe protein contained 26 ± 4 Fe atoms; it did not contain Mo, V, or any other heterometal atom.

The most significant catalytic property of the iron‐only nitrogenase is its high H2‐producing activity, which is much less inhibited by competitive substrates than the activity of the conventional molybdenum nitrogenase. Under optimal conditions for N2 reduction, the activity ratios (mol N2 reduced/mol H2 produced) obtained were 1:1 (molybdenum nitrogenase) and 1:7.5 (iron nitrogenase). The Rc1Fe protein has only a very low affinity for C2H2. The Km value determined (12.5 kPa), was about ninefold higher than the Km for Rc1Mo (1.4kPa). The proportion of ethane produced from acetylene (catalyzed by the iron nitrogenase), was strictly pH dependent. It corresponded to 5.5% of the amount of ethylene at pH 6.5 and was almost zero at pH values greater than 8.5.

In complementation experiments, component 1 proteins coupled very poorly with the ‘wrong’ component 2. Rc1Fe, if complemented with Rc2Mo, showed only 10–15% of the maximally possible activity. Cross‐reaction experiments with isolated polyclonal antibodies revealed that Rc1Fe and Rc1Mo are immunologically not related.

The most active Rc1Fe samples appeared to be EPR‐silent in the Na2S2O4‐reduced state. However, on partial oxidation with K3[Fe(CN)6] or thionine several signals occurred. The most significant signal appears to be the one at g= 2.27 and 2.06 which deviates from all signals so far described for P clusters. It is a transient signal that appears and disappears reversibly in a redox potential region between ‐100 mV and +150 mV. Another novel EPR signal (g= 1.96, 1.92, 1.77) occurred on further reduction of Rc1Fe by using turnover conditions in the presence of a substrate (N2, C2H2, H+).
Further Characterization of a Rat Hepatoma‐Derived Aldose‐Reductase‐Like Protein
Authors: Zeindl‐Eberhart Evelyn , Jungblut Peter R. , Otto Albrecht , Kerler Rosi , Rabes Hartmut M.
Published: July 2004

A protein detected in N‐methyl‐N‐nitrosourea‐initiated rat hepatomas by two‐dimensional electrophoresis at 35 kDd/pI 7.4 was identified in a previous study by internal amino acid micro sequencing as an aldose‐reductase‐like protein [Zeindl‐Eberhart, E., Jungblut, P. R., Otto, A. & Rabes, H. M. (1994) Identification of tumor‐associated protein variants during rat hepatocarcinogenesis, J. Biol. Chem. 269, 14589–145941. Two‐dimensional electrophoresis of rat lens proteins revealed a spot at 37 kDa/pI 6.8 that showed a high degree of identity (98.5%) with rat lens aldose reductase after amino acid sequencing and 80% sequence identity to the rat‐hepatoma‐derived aldose‐reductase‐like protein. This suggests that hepatoma‐derived aldose‐reductase‐like protein and rat lens aldose reductase are related proteins encoded by different genes. A different expression profile of these proteins was found in various rat organs. Rat lens aldose reductase is present, in addition to in lens, in heart, brain, muscle, lung, duodenum, kidney, spleen and bone marrow, while the hepatoma‐derived aldose‐reductase‐like protein is found preferentially in hepatomas and in embryonic liver. Though different in organ expression, an identical response was found for both proteins after stimulation with fibroblast growth factor‐1 and after exposure to increased glucose concentrations. Since rat hepatoma‐derived aldose‐reductase‐like protein is expressed in embryonic, but not in adult liver, it is assumed that it is expressed in hepatomas as a functionally active embryonal type of aldose reductase during hepatocarcinogenesis. Immunohistochemistry revealed that the hepatoma‐derived aldose‐reductase‐like protein is expressed already in the preneoplastic stage of hepatocarcinogenesis and might potentially serve as a marker enzyme in early hepatic neoplasia.
Translocation to the Periplasm and Signal Sequence Cleavage of Preapocytochrome c Depend on Sec and Lep, but not on the ccm gene products
Authors: Thöny‐Meyer Linda , Künzler Peter
Published: July 2004

Post‐translational maturation of soluble cytochrome c includes translocation of the precursor polypeptide and heme through the cytoplasmic membrane, proteolytic cleavage of the signal sequence, and cova‐lent attachment of heme. Specific genes for cytochrome c maturation (ccmABCDEFGH in Escherichia coli) are required for holocytochrome c formation, among them genes encoding an ABC transporter (ccmABC). We investigated the requirements of apocytochrome translocation to the periplasm and characterized specific intermediates of the cytochrome c maturation pathway. Apocytochrome precursor was present in the membrane fraction. Translocation of the polypeptide was independent of ccm gene products, but dependent on a functional secretion machinery, as shown by accumulation of preapocytochrome c in the membranes of secA and secY mutants. After translocation, cleavage of the signal sequence allowed the release of apocytochrome into the periplasm, where heme was bound in a ccm‐dependent manner. By contrast, non‐cleaved holocytochrome c containing covalently bound heme accumulated in the membranes of a lepB mutant, which indicated that signal sequence cleavage and heme attachment are independent steps in the cytochrome c maturation pathway.