bluuebunny/crossref_metadata_2025 huggingface dataset:
Interactions of the C‐terminus of viral protein R with nucleic acids are modulated by its N‐terminus
Authors: de Rocquigny Hugues , Caneparo Anne , Delaunay Thierry , Bischerour Julien , Mouscadet Jean‐François , Roques Bernard P.
Published: March 2003

The basic viral protein R (Vpr) performs several functions during the human immunodeficiency virus HIV‐1 retroviral cycle, including G2 mitosis arrest and nuclear import of the preintegration complex allowing lentivirus to replicate in nondividing cells. Accordingly, this protein was found in the nucleus of infected cells. In the virus, Vpr is incorporated through interaction with both nucleocapsid protein 7 (NCp7) and p6, two small proteins encoded by the C‐terminal part of the Gag precursor. NCp7 is also involved in genomic RNA encapsidation during the budding process suggesting a possible interaction of Vpr with nucleic acids, either directly or via the NCp7 intermediate. Gel shift experiments were carried out with RNA and DNA using synthetic Vpr and peptide derivatives. The results show that Vpr binds to nucleic‐acid inducing aggregates. This process, which requires the C‐terminal basic domain of the protein (in particular the helical 70–80 domain), is regulated by the N‐terminal region of Vpr. Moreover, NCp7 was shown to enhance RNA recognition by Vpr, a feature that could be required for Vpr encapsidation and during nuclear import of the preintegration complex.
Characterization of a second methylene tetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1
Authors: Hagemeier Christoph H. , Chistoserdova Ludmila , Lidstrom Mary E. , Thauer Rudolf K. , Vorholt Julia A.
Published: March 2003

Cell extracts of Methylobacterium extorquens AM1 were recently found to catalyze the dehydrogenation of methylene tetrahydromethanopterin (methylene H4MPT) with NAD+ and NADP+. The purification of a 32‐kDa NADP‐specific methylene H4MPT dehydrogenase (MtdA) was described already. Here we report on the characterization of a second methylene H4MPT dehydrogenase (MtdB) from this aerobic α‐proteobacterium. Purified MtdB with an apparent molecular mass of 32 kDa was shown to catalyze the oxidation of methylene H4MPT to methenyl H4MPT with NAD+ and NADP+ via a ternary complex catalytic mechanism. The Km for methylene H4MPT was 50 µm with NAD+ (Vmax = 1100 U·mg−1) and 100 µm with NADP+ (Vmax = 950 U·mg−1). The Km value for NAD+ was 200 µm and for NADP+ 20 µm. In contrast to MtdA, MtdB could not catalyze the dehydrogenation of methylene tetrahydrofolate. Via the N‐terminal amino‐acid sequence, the MtdB encoding gene was identified to be orfX located in a cluster of genes whose translated products show high sequence identities to enzymes previously found only in methanogenic and sulfate reducing archaea. Despite its location, MtdB did not show sequence similarity to archaeal enzymes. The highest similarity was to MtdA, whose encoding gene is located outside of the archaeal island. Mutants defective in MtdB were unable to grow on methanol and showed a pronounced sensitivity towards formaldehyde. On the basis of the mutant phenotype and of the kinetic properties, possible functions of MtdB and MtdA are discussed. We also report that both MtdB and MtdA can be heterologously overproduced in Escherichia coli making these two enzymes readily available for structural analysis.
A bifunctional Δ6‐fatty acyl acetylenase/desaturase from the moss Ceratodon purpureus
Authors: Sperling Petra , Lee Michael , Girke Thomas , Zähringer Ulrich , Stymne Sten , Heinz Ernst
Published: March 2003

Many plant genes have been cloned that encode regioselective desaturases catalyzing the formation of cis‐unsaturated fatty acids. However, very few genes have been cloned that encode enzymes catalyzing the formation of the functional groups found in unusual fatty acids (e.g. hydroxy, epoxy or acetylenic fatty acids). Here, we describe the characterization of an acetylenase from the moss Ceratodon purpureus with a regioselectivity differing from the previously described Δ12‐acetylenase. The gene encoding this protein, together with a Δ6‐desaturase, was cloned by a PCR‐based approach with primers derived from conserved regions in Δ5‐, Δ6‐fatty‐acid desaturases and Δ8‐sphingolipid desaturases. The proteins that are encoded by the two cloned cDNAs are likely to consist of a N‐terminal extension of unknown function, a cytochrome b5‐domain, and a C‐terminal domain that is similar to acyl lipid desaturases with characteristic histidine boxes. The proteins were highly homologous in sequence to the Δ6‐desaturase from the moss Physcomitrella patens. When these two cDNAs were expressed in Saccharomyces cerevisiae, both transgenic yeast cultures desaturated Δ9‐unsaturated C16‐ and C18‐fatty acids by inserting an additional Δ6cis‐double bond. One of these transgenic yeast clones was also able to introduce a Δ6‐triple bond into γ‐linolenic and stearidonic acid. This resulted in the formation of 9,12,15‐(Z,Z,Z)‐octadecatrien‐6‐ynoic acid, the main fatty acid found in C. pupureus. These results demonstrate that the Δ6‐acetylenase from C. pupureus is a bifunctional enzyme, which can introduce a Δ6cis‐double bond into 9,12,(15)‐C18‐polyenoic acids as well as converting a Δ6cis‐double bond to a Δ6‐triple bond.
The involvement of coenzyme A esters in the dehydration of (R)‐phenyllactate to (E)‐cinnamate by Clostridium sporogenes
Authors: Dickert Sandra , Pierik Antonio J. , Linder Dietmar , Buckel Wolfgang
Published: March 2003

Phenyllactate dehydratase from Clostridium sporogenes grown anaerobically on l‐phenylalanine catalyses the reversible syn‐dehydration of (R)‐phenyllactate to (E)‐cinnamate. Purification yielded a heterotrimeric enzyme complex (130 ± 15 kDa) composed of FldA (46 kDa), FldB (43 kDa) and FldC (40 kDa). By re‐chromatography on Q‐Sepharose, the major part of FldA could be separated and identified as oxygen insensitive cinnamoyl‐CoA:phenyllactate CoA‐transferase, whereas the transferase depleted trimeric complex retained oxygen sensitive phenyllactate dehydratase activity and contained about one [4Fe‐4S] cluster. The dehydratase activity required 10 µm FAD, 0.4 mm ATP, 2.5 mm MgCl2, 0.1 mm NADH, 5 µm cinnamoyl‐CoA and small amounts of cell‐free extract (10 µg protein per mL) similar to that known for 2‐hydroxyglutaryl‐CoA dehydratase from Acidaminococcus fermentans. The N‐terminus of the homogenous FldA (39 amino acids) is homologous to that of CaiB (39% sequence identity) involved in carnitine metabolism in Escherichia coli. Both enzymes are members of an emerging group of CoA‐transferases which exhibit high substrate specificity but apparently do not form enzyme CoA‐ester intermediates. It is concluded that dehydration of (R)‐phenyllactate to (E)‐cinnamate proceeds in two steps, a CoA‐transfer from cinnamoyl‐CoA to phenyllactate, catalysed by FldA, followed by the dehydration of phenyllactyl‐CoA, catalysed by FldB and FldC, whereby the noncovalently bound prosthetic group cinnamoyl‐CoA is regenerated. This demonstrates the necessity of a 2‐hydroxyacyl‐CoA intermediate in the dehydration of 2‐hydroxyacids. The transient CoA‐ester formation during the dehydration of phenyllactate resembles that during citrate cleavage catalysed by bacterial citrate lyase, which contain a derivative of acetyl‐CoA covalently bound to an acyl‐carrier‐protein (ACP).
Atrans‐acting factor, isolated by the three‐hybrid system, that influences alternative splicing of the amyloid precursor protein minigene
Authors: Poleev Andrej , Hartmann Annette , Stamm Stefan
Published: March 2003

Two clones were isolated in a three‐hybrid screen of a rat fetal brain P5 cDNA library with an intronic splicing enhancer of the amyloid precursor protein (APP) gene as RNA bait. These clones represent the rat homologues of the previously described genes CUG‐binding protein (CUG‐BP) and Siah‐binding protein (Siah‐BP). Both interact in a sequence‐specific manner with the RNA bait used for library screening as well as with the CUG repeat. In contrast, no interactions were observed in the three‐hybrid assay with other baits tested. In two‐hybrid assays, Siah‐BP interacts with U2AF65 as well as with itself. EWS, an RGG‐type RNA‐binding protein associated with Ewing sarcoma, was identified as an interacting partner for the CUG‐BP homologue in a two‐hybrid assay for protein–protein interactions performed with various factors involved in RNA metabolism. Splicing assays performed by RT‐PCR from cells cotransfected with certain cDNAs and an APP minigene, used as a reporter, indicate exclusion of exon 8 if the CUG‐BP homologue is present. We conclude that clone AF169013 and its counterpart in human CUG‐BP could be thetrans‐acting factors that interact with the splicing enhancer downstream of exon 8, and in this way influence alternative splicing of the APP minigene.
Phytanoyl‐CoA hydroxylase activity is induced by phytanic acid
Authors: Zomer Anna W. M. , Jansen Gerbert A. , van der Burg Bart , Verhoeven Nanda M. , Jakobs Cornelis , van der Saag Paul T. , Wanders Ronald J. A. , Poll‐The Bwee Tien
Published: March 2003

Phytanic acid (3,7,11,15‐tetramethylhexadecanoic acid) is a branched‐chain fatty acid present in various dietary products such as milk, cheese and fish. In patients with Refsum disease, accumulation of phytanic acid occurs due to a deficiency of phytanoyl‐CoA hydroxylase, a peroxisomal enzyme containing a peroxisomal targeting signal 2. Recently, phytanoyl‐CoA hydroxylase cDNA has been isolated and functional mutations have been identified. As it has been shown that phytanic acid activates the nuclear hormone receptors peroxisome proliferator‐activated receptor (PPAR)α and all three retinoid X receptors (RXRs), the intracellular concentration of this fatty acid should be tightly regulated.

When various cell lines were grown in the presence of phytanic acid, the activity of phytanoyl‐CoA hydroxylase increased up to four times, depending on the particular cell type. In one cell line, HepG2, no induction of phytanoyl‐CoA hydroxylase activity was observed. After addition of phytanic acid to COS‐1 cells, an increase in phytanoyl‐CoA hydroxylase activity was observed within 2 h, indicating a quick cell response. No stimulation of phytanoyl‐CoA hydroxylase was observed when COS‐1 cells were grown in the presence of clofibric acid, 9‐cis‐retinoic acid or both ligands together. This indicates that the activation of phytanoyl‐CoA hydroxylase is not regulated via PPARα or RXR. However, stimulation of PPARα and all RXRs by clofibric acid and 9‐cis‐retinoic acid was observed in transient transfection assays. These results suggest that the induction of phytanoyl‐CoA hydroxylase by phytanic acid does not proceed via one of the nuclear hormone receptors, RXR or PPARα.
Analysis of CAK activities from human cells
Authors: Kaldis Philipp , Solomon Mark J.
Published: March 2003

The cdk‐activating kinase (CAK) activates cyclin‐dependent kinases (cdks) that control cell‐cycle progression by phosphorylating a threonine residue conserved in cdks. CAK from humans contains p40MO15 (cdk7), cyclin H and MAT1, which are also subunits of transcription factor IIH where they phosphorylate the C‐terminal domain of the large subunit of RNA polymerase II. In contrast, budding yeast Cak1p is a monomeric enzyme without C‐terminal domain kinase activity. Here, we analyze CAK activities in HeLa cells using cdk2‐affinity chromatography. In addition to MO15, a second CAK activity was detected that runs on gel filtration at 30–40 kDa. This activity phosphorylated and activated cdk2 and cdk6. Furthermore, this ‘small CAK’ activity resembled Cak1p rather than MO15 in terms of substrate specificity, reactivity to antibodies against MO15 and Cak1p, and sensitivity to 5′‐fluorosulfonylbenzoyladenosine, an irreversible inhibitory ATP analog. Our findings suggest the presence of at least two different CAK activities in human cells.
Fluorescent microplate assay for cancer cell‐associated cathepsin B
Authors: Hulkower Keren I. , Butler Christopher C. , Linebaugh Bruce E. , Klaus Jeffrey L. , Keppler Daniel , Giranda Vincent L. , Sloane Bonnie F.
Published: March 2003

Cathepsin B and in particular cell‐surface and secreted cathepsin B has been implicated in the invasive and metastatic phenotype of numerous types of cancer. We describe here a method to easily survey cancer cell lines for cathepsin B activity using the highly selective substrate Z‐Arg‐Arg‐AMC. Intact human U87 glioma cells hydrolyze Z‐Arg‐Arg‐AMC with a Km of 460 µm at pH 7.0 and 37 °C. This is nearly the same as the Km of 430 µm obtained with purified cathepsin B assayed under the same conditions. The pericellular (i.e. both cell‐surface and released) cathepsin B activity was inhibited by the cysteine protease inhibitors E‐64, leupeptin, Mu‐Np2‐HphVS‐2Np, Mu‐Leu‐HpHVSPh and the cathepsin B selective inhibitor Mu‐Tyr(3,5 I2)‐HphVSPh with IC50 values similar to those observed for the inhibition of purified human liver cathepsin B. Other human cancer cell lines with measurable pericellular cathepsin B activity included HT‐1080 fibrosarcoma, MiaPaCa pancreatic, PC‐3 prostate and HCT‐116 colon. Cathepsin B activity correlated with protein levels of cathepsin B as determined by immunoblot analysis. Pericellular cathepsin B activity was also detected in the rat cell lines MatLyLu prostate and Mat B III adenocarcinoma and in the murine lines B16a melanoma and Lewis lung carcinoma. The ability to determine pericellular cathepsin B activity will be useful in selecting appropriate cell lines for use in vivo when analyzing the effects of inhibiting cathepsin B activity on tumor growth and metastasis.
Purification and characterization of cytosolic pyruvate kinase from Brassica napus (rapeseed) suspension cell cultures
Authors: Smith Christopher R. , Knowles Vicki L. , Plaxton William C.
Published: March 2003

Cytosolic pyruvate kinase (PKc) from Brassica napus suspension cells was purified 201‐fold to electrophoretic homogeneity and a final specific activity of 51 µmol phosphoenolpyruvate utilized per min per mg protein. SDS/PAGE and gel filtration analyses of the final preparation indicated that this PKc is a 220‐kDa homotetramer composed of 56‐kDa subunits. The enzyme was relatively heat‐stable and displayed a broad pH optimum of pH 6.8. PKc activity was absolutely dependent upon the simultaneous presence of a bivalent and univalent cation, with Mg2+ and K+ fulfilling this requirement. Hyperbolic saturation kinetics were observed for phosphoenolpyruvate, ADP, Mg2+ and K+ (apparent Km values = 0.12, 0.075, 0.21 and 0.48 mm, respectively). Although the enzyme utilized UDP, CDP and IDP as alternative nucleotides, ADP was the preferred substrate. l‐Glutamate, oxalate, and the flavonoids rutin and quercetin were the most effective inhibitors (I50 values = 4, 0.3, 0.07, and 0.10 mm, respectively).l‐Aspartate functioned as an activator (Ka = 0.31 mm) by causing a 40% increase in Vmax while completely reversing the inhibition of PKc by l‐glutamate. Reciprocal control by l‐aspartate and l‐glutamate is specific for these amino acids and provides a rationale for the in vivo activation of PKc that occurs during periods of enhanced NH +4‐assimilation. Allosteric features of B. napus PKc are compared with those of B. napus phosphoenolpyruvate carboxylase. A model is presented that highlights the pivotal role of l‐aspartate and l‐glutamate in the coordinate regulation of these key phosphoenolpyruvate utilizing cytosolic enzymes.
Expression of immediate early gene pip92 during anisomycin‐induced cell death is mediated by the JNK‐ and p38‐dependent activation of Elk1
Authors: Chung Kwang C. , Kim Sung M. , Rhang Sungin , Lau Lester F. , Gomes Ignatius , Ahn Young S.
Published: March 2003

We report here that immediate early gene pip92 is expressed during anisomycin‐induced cell death in fibroblast NIH3T3 cells. To determine the mechanism by which this occurs and to identify downstream signaling pathways, we investigated the induction of the pip92 promoter. The activation of pip92 by anisomycin is mediated by the activation of MAP kinases, such as JNK and p38 kinase, but not ERK. Deletion analysis of the pip92 promoter indicated that pip92 activation occurs primarily within the region containing a serum response element (SRE). Further analysis of the SRE using a heterologous thymidine kinase promoter showed that both an Ets and CArG‐like site are required for anisomycin‐induced pip92 expression. Elk1, which binds to the Ets site, was phosphorylated by the JNK‐ and p38‐dependent pathways and the phosphorylation of Elk1–GAL4 fusion proteins by these pathways was sufficient for the transactivation. Overall, this study suggested that different MAPK pathways are involved in the expression of immediate early gene pip92 by growth factors and environmental stresses.
Annexin V relocates to the platelet cytoskeleton upon activation and binds to a specific isoform of actin
Authors: Tzima Eleni , Trotter Patrick J. , Orchard Margaret A. , Walker John H.
Published: March 2003

We have previously reported that stimulation of platelets causes a relocation of annexin V to the cytoplasmic side of the plasma membrane where it associates with actin. This study examined the association of annexin V with the platelet cytoskeleton and its binding to actin, following both physiological activation with thrombin and Ca2+‐ionophore activation. The time‐dependence of annexin V incorporation into the detergent‐extracted cytoskeleton following activation with thrombin was also measured. Although calcium from the intracellular stores was enough to relocate intracellular annexin V to the cytoskeleton, this relocation was further enhanced by influx of extracellular calcium. The association of annexin V with the cytoskeleton was found to be unaffected by the action of cytochalasin E, however, annexin V was solubilized when DNase I was used to depolymerize the membrane cytoskeleton, and spontaneously re‐associated with the actin filaments when re‐polymerization was inducedin vitro. Using a bifunctional crosslinking reagent we have identified an 85‐kDa complex in both membrane and cytoskeleton fractions containing annexin V and actin. Direct binding to actin filaments was only observed in high [Ca2+], however, inclusion of an extract from thrombin‐stimulated platelets lowered the [Ca2+] requirement for the binding of annexin V to F‐actin to physiological levels. We also show that GST–annexin V mimics the physiological binding of annexin V to membranes, and that this GST–annexin V binds directly to a specific isoform of actin. Immunoprecipitation using antibodies against annexin V copurify annexin V and γ‐ but not β‐actin from activated platelets. This is the first report of a possible preferential binding of annexin V to a specific isoform of actin, namely γ‐actin. The results of this study suggest a model in which annexin V that relocates to the plasma membrane and binds to γ‐actin in an activation‐dependent manner forms a strong association with the platelet cytoskeleton.
Purification, characterization and amino‐acid sequence analysis of a thermostable, low molecular mass endo‐β‐1,4‐glucanase from blue mussel, Mytilus edulis
Authors: Xu Bingze , Hellman Ulf , Ersson Bo , Janson Jan‐Christer
Published: March 2003

A cellulase (endo‐β‐1,4‐d‐glucanase, EC 3.2.1.4) from blue mussel (Mytilus edulis) was purified to homogeneity using a combination of acid precipitation, heat precipitation, immobilized metal ion affinity chromatography, size‐exclusion chromatography and ion‐exchange chromatography. Purity was analyzed by SDS/PAGE, IEF and RP‐HPLC. The cellulase (endoglucanase) was characterized with regard to enzymatic properties, isoelectric point, molecular mass and amino‐acid sequence. It is a single polypeptide chain of 181 amino acids cross‐linked with six disulfide bridges. Its molecular mass, as measured by MALDI‐MS, is 19 702 Da; a value of 19 710.57 Da was calculated from amino‐acid composition. The isoelectric point of the enzyme was estimated by isoelectric focusing in a polyacrylamide gel to a value of 7.6. According to amino‐acid composition, the theoretical pI is 7.011. The effect of temperature on the endoglucanase activity, with carboxymethyl cellulose and amorphous cellulose as substrates, respectively, was studied at pH 5.5 and displayed an unusually broad optimum activity temperature range between 30 and 50 °C. Another unusual feature is that the enzyme retains 55–60% of its maximum activity at 0 °C. The enzyme readily degrades amorphous cellulose and carboxymethyl cellulose but displays no hydrolytic activity towards crystalline cellulose (Avicel) and shows no cross‐specificity for xylan; there is no binding to Avicel. The enzyme can withstand 10 min at 100 °C without irreversible loss of enzymatic activity. Amino‐acid sequence‐based classification has revealed that the enzyme belongs to the glycoside hydrolase family 45, subfamily 2 (B. Henrissat, Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France, personal communication).
Amino‐acid replacements in an internal region of tropomyosin alter the properties of the entire molecule
Authors: Sano Ken‐Ichi , Maeda Kayo , Taniguchi Hisaaki , Maéda Yuichiro
Published: March 2003

Two isoforms of lobster muscle tropomyosin, a fast muscle type, fTm, and a slow muscle type, sTm1, are identical except for 15 residues within the region of amino acids 39–80, which corresponds to exon 2 of the tropomyosin genes of many phyla. Although the difference in the sequence does not include the terminal regions, the two isoforms are extremely different in viscosity, which is a good measure of the head‐to‐tail interaction strength and should be dependent on the conformation of the terminal 7–9 residues. To determine the influence of amino‐acid replacements in the internal region on the overall conformation and the functional properties of the molecule, we compared the physical properties of the two isoforms and their interactions with other proteins, such as actin and myosin subfragment 1 (S1). Limited proteolysis by trypsin and chymotrypsin showed that sTm1 is more susceptible than fTm at the sites outside the region with the replaced residues. Compared with fTm, sTm1 showed higher viscosity, had a higher actin affinity, and inhibited acto‐S1 ATPase to a greater extent. Finally, the binding isotherm of S1‐ADP to actin‐sTm1 is less sigmoidal than that to actin‐fTm. These results indicate that the amino‐acid replacements in the internal region alter the conformation and the physical properties of the entire molecule as well as its interactions with actin and myosin.
Highly potent inhibitors of human cathepsin L identified by screening combinatorial pentapeptide amide collections
Authors: Brinker Achim , Weber Ekkehard , Stoll Dieter , Voigt Jürgen , Müller Annett , Sewald Norbert , Jung Günther , Wiesmüller Karl‐Heinz , Bohley Peter
Published: March 2003

By screening a combinatorial pentapeptide amide collection in an inhibition assay, we systematically evaluated the potential of 19 proteinogenic amino acids and seven nonproteinogenic amino acids to serve as building blocks for inhibitors of human cathepsin L. Particularly efficient were aromatic, bulky, hydrophobic amino‐acid residues, especially leucine, and positively charged residues, especially arginine. Building blocks for potential inhibitory peptides were combined by random selection from their activity pattern. This random approach for the design of inhibitors was introduced to compensate for the inaccuracy induced by shifted docking of combinatorial compound collections at the active center of cathepsin L. Thereby, we obtained structurally defined pentapeptide amides which inhibited human cathepsin L at nanomolar concentrations. Among the most potent novel inhibitors, one peptide, RKLLW‐NH2, shares the amphiphilic character of the nonamer fragment VMNGLQNRK of the autoinhibitory, substrate‐like, but reverse‐binding prosegment of human cathepsin L which blocks the active center of the enzyme. Obviously, RKLLW‐NH2 carries the functions that are important for enzyme–peptide interaction in a condensed form. This hypothesis was confirmed by structure–activity studies using truncated and modified pentapeptides.
The crystal structure of the complex of Zea maysα subunit with a fragment of human β subunit provides the clue to the architecture of protein kinase CK2 holoenzyme
Authors: Battistutta Roberto , Sarno Stefania , De Moliner Erika , Marin Oriano , Issinger Olaf‐G. , Zanotti Giuseppe , Pinna Lorenzo A.
Published: March 2003

The crystal structure of a complex between the catalytic α subunit of Zea mays CK2 and a 23‐mer peptide corresponding the C‐terminal sequence 181–203 of the human CK2 regulatory β subunit has been determined at 3.16‐Å resolution. The complex, composed of two α chains and two peptides, presents a molecular twofold axis, with each peptide interacting with both α chains. In the derived model of the holoenzyme, the regulatory subunits are positioned on the opposite side with respect to the opening of the catalytic sites, that remain accessible to substrates and cosubstrates. The β subunit can influence the catalytic activity both directly and by promoting the formation of the α2 dimer, in which each α chain interacts with the active site of the other. Furthermore, the two active sites are so close in space that they can simultaneously bind and phosphorylate two phosphoacceptor residues of the same substrate.
Mitochondrial and submitochondrial localization of human deoxyguanosine kinase
Authors: Jüllig Mia , Eriksson Staffan
Published: March 2003

Deoxyguanosine kinase and thymidine kinase 2 are responsible for catalysing the first step in the salvage of deoxynucleosides in mitochondria. These enzymes also play an important role in activating several antiviral and anticancer nucleoside analogs, which may lead to unwanted side‐effects when the resulting nucleotides are incorporated into the mitochondrial genome. We studied deoxyguanosine kinase in submitochondrial fractions from human placental mitochondria. It was localized in the mitochondrial matrix fraction by Western blotting using a purified polyclonal antibody. This antibody was also used in an immunohistochemical in situ experiment with human embryonic kidney 293 cells, in which the deoxyguanosine kinase antibody colocalized with a mitochondrion‐specific fluorescent probe and there was no significant cytosolic staining.
Functional regions in the essential light chain of smooth muscle myosin as revealed by the mutagenesis approach
Authors: Quevillon‐Chéruel Sophie , Janmot Chantal , Nozais Muriel , Lompré Anne‐Marie , Béchet Jean‐Jacques
Published: March 2003

The endogenous essential light chain (LC17) of myosin from intestine smooth muscle was replaced with mutated essential light chains prepared using recombinant techniques. Complete exchange was observed with histidine‐tagged derivatives of LC17a, LC17b and E122A‐LC17a (LC17a and LC17b are the usual constituants of smooth muscle myosin), with small changes in the ATPase activity of reconstituted myosins. Much less exchange was observed with the light‐chain derivative lacking the last 12 amino acid residues, demonstrating the importance of this segment, which may act as one arm of a pair of pincers to bind the myosin heavy chain.
AhpF and other NADH:peroxiredoxin oxidoreductases, homologues of low Mr thioredoxin reductase
Authors: Poole Leslie B. , Reynolds C. Michael , Wood Zachary A. , Karplus P. Andrew , Ellis Holly R. , Li Calzi Marco
Published: March 2003

A group of bacterial flavoproteins related to thioredoxin reductase contain an additional ≈ 200‐amino‐acid domain including a redox‐active disulfide center at their N‐termini. These flavoproteins, designated NADH:peroxiredoxin oxidoreductases, catalyze the pyridine‐nucleotide‐dependent reduction of cysteine‐based peroxidases (e.g. Salmonella typhimurium AhpC, a member of the peroxiredoxin family) which in turn reduce H2O2 or organic hydroperoxides. These enzymes catalyze rapid electron transfer (kcat > 165 s−1) through one tightly bound FAD and two redox‐active disulfide centers, with the N‐terminal‐most disulfide center acting as a redox mediator between the thioredoxin‐reductase‐like part of these proteins and the peroxiredoxin substrates. A chimeric protein with the first 207 amino acids of S. typhimurium AhpF attached to the N‐terminus of Escherichia coli thioredoxin reductase exhibits very high NADPH:peroxiredoxin oxidoreductase and thioredoxin reductase activities. Catalytic turnover by NADH:peroxiredoxin oxidoreductases may involve major domain rotations, analogous to those proposed for bacterial thioredoxin reductase, and cycling of these enzymes between two electron‐reduced (EH2) and four electron‐reduced (EH4) redox states.
Catalytic differences between porcine blastocyst and placental aromatase isozymes
Authors: Kao Y.‐C. , Higashiyama T. , Sun X. , Okubo T. , Yarborough C. , Choi I. , Osawa Y. , Simmen F. A. , Chen S.
Published: March 2003

Two isozymes of porcine aromatase, the placental and the blastocyst forms, were expressed in CHO cells using the mammalian cell transfection method. Using an ‘in‐cell’ assay (a 3H‐water release method), catalytic parameters of the porcine placental aromatase were found to be very similar to those of the human enzyme; however, the activity of the blastocyst isozyme was found to be one‐thirtieth that of the placental isozyme. Product isolation assay (using testosterone as the substrate) revealed that the major steroid products were 17β‐estradiol and 19‐nortestosterone. The product ratio of estradiol/19‐nortestosterone was found to be 94 : 6 for the porcine placental form, 6 : 94 for the porcine blastocyst form, and 92 : 8 for the human wild‐type aromatase. Therefore, the porcine blastocyst aromatase isozyme catalyzes mainly androgen 19‐desmethylation rather than aromatization. In addition, inhibition profile analyses on the placental and blastocyst isozymes were performed using three steroidal inhibitors [4‐hydroxyandro‐stenedione (4‐OHA), 7α‐(4′‐amino)phenylthio‐1,4‐androstandiene‐3,17‐dione (7α‐APTADD), and bridge (2,19‐methyleneoxy) androstene‐3,17‐dione (MDL 101,003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)]. While the two isozymes of porcine aromatase share 93% amino‐acid sequence identity, our results indicate that the two porcine aromatase isozymes have distinct responses to various aromatase inhibitors.
Identification of neoxanthin synthase as a carotenoid cyclase paralog
Authors: Bouvier Florence , D’Harlingue Alain , Backhaus Ralph A. , Kumagai Monto H. , Camara Bilal
Published: March 2003

Neoxanthin, a precursor of the plant hormone abscisic acid, is an allenic xanthophyll recognized as the last product of carotenoid synthesis in green plants. A cDNA for neoxanthin synthase (NSY) was isolated from tomato using a molecular approach based on the mechanistic and structural similarities of NSY to two other closely related carotenogenic enzymes, lycopene cyclase (LCY) and capsanthin‐capsorubin synthase (CCS). The identified tomato NSY cDNA (T.NSY) encodes a 56‐kDa plastid‐targeted protein that when expressed in Escherichia coli, catalyzes the conversion of violaxanthin to neoxanthin. In tobacco leaves that transiently express T.NSY, an increase in neoxanthin content with a concomitant decrease in violaxanthin is observed. NSY is structurally similar to LCY and CCS. However, in Cyanobacteria, the generally accepted progenitor of plastids, both CCS and NSY are absent while LCY is present. LCY catalyzes a simplified version of the reaction catalyzed by NSY and CCS suggesting that these two enzymes were remodeled from LCY during higher plant evolution to create new forms of oxidized carotenoids.
Heme‐(hydro)peroxide mediated O‐ and N‐dealkylation
Authors: Boersma Marelle G. , Primus Jean‐Louis , Koerts Janneke , Veeger Cees , Rietjens Ivonne M. C. M.
Published: March 2003

The mechanism of microperoxidase‐8 (MP‐8) mediated O‐ and N‐dealkylation was investigated. In the absence of ascorbate (peroxidase mode), many unidentified polymeric products are formed and the extent of substrate degradation correlates (r = 0.94) with the calculated substrate ionization potential, reflecting the formation of radical intermediates.

In the presence of ascorbate (P450 mode) formation of polymeric products is largely prevented but, surprisingly, dealkylation is not affected. In addition, aromatic hydroxylation and oxidative dehalogenation is observed. The results exclude a radical mechanism and indicate the involvement of a (hydro)peroxo‐iron heme intermediate in P450‐type of heteroatom dealkylation.
Novel α‐L‐fucosidase inhibitors from the bark of Angylocalyx pynaertii (Leguminosae)
Authors: Asano Naoki , Yasuda Kayo , Kizu Haruhisa , Kato Atsushi , Fan Jian‐Qiang , Nash Robert J. , Fleet George W. J. , Molyneux Russell J.
Published: March 2003

The extract of bark of Angylocalyx pynaertii (Leguminosae) was found to potently inhibit mammalian α‐
l‐fucosidases. A thorough examination of the extract resulted in the discovery of 15 polyhydroxylated alkaloids, including the known alkaloids from seeds of this plant, 1,4‐dideoxy‐1,4‐imino‐
d‐arabinitol (DAB), 1‐deoxymannojirimycin (DMJ) and 2,5‐imino‐1,2,5‐trideoxy‐
d‐mannitol (6‐deoxy‐DMDP). Among them, eight sugar‐mimic alkaloids showed the potent inhibitory activity towards bovine epididymis α‐
l‐fucosidase and their Ki values are as follows: 6‐deoxy‐DMDP (83 µm), 2,5‐imino‐1,2,5‐trideoxy‐
l‐glucitol (0.49 µm), 2,5‐dideoxy‐2,5‐imino‐
d‐fucitol (17 µm), 2,5‐imino‐1,2,5‐trideoxy‐
d‐altritol (3.7 µm), DMJ (4.7 µm), N‐methyl‐DMJ (30 µm), 6‐O‐α‐
l‐rhamnopyranosyl‐DMJ (Rha‐DMJ, 0.06 µm), and β‐
l‐homofuconojirimycin (β‐HFJ, 0.0053 µm). We definitively deduced the structural requirements of inhibitors of α‐
l‐fucosidase for the piperidine alkaloids (DMJ derivatives). The minimum structural feature of α‐
l‐fucosidase inhibitors is the correct configuration of the three hydroxyl groups on the piperidine ring corresponding to C2, C3 and C4 of
l‐fucose. Furthermore, the addition of a methyl group in the correct configuration to the ring carbon atom corresponding to C5 of
l‐fucose generates extremely powerful inhibition of α‐
l‐fucosidase. The replacement of the methyl group of β‐HFJ by a hydroxymethyl group reduced its inhibitory potential about 80‐fold. This suggests that there may be a hydrophobic region in or around the active site. The existence or configuration of a substituent group on the ring carbon atom corresponding to the anomeric position of
l‐fucose does not appear to be important for the inhibition. Interestingly, Rha‐DMJ was a 70‐fold more potent inhibitor of α‐
l‐fucosidase than DMJ. This implies that the lysosomal α‐
l‐fucosidase may have subsites recognizing oligosaccharyl structures in natural substrates.
Scots pine expresses short‐root‐specific peroxidases during development
Authors: Tarkka Mika T. , Nyman Tuula A. , Kalkkinen Nisse , Raudaskoski Marjatta
Published: March 2003

Nine short‐root‐specific proteins from Scots pine (Pinus sylvestris L.) detected and isolated as individual spots by 2D‐PAGE were identified. The similar peptide mass maps obtained for all nine polypeptide spots together with lectin‐blotting results suggest that they represent forms of the same modified protein. N‐Terminal sequence analysis of two of the peptides showed high similarity to peroxidases. RT‐PCR with oligonucleotide primers corresponding to determined peptide sequences and conserved regions in plant peroxidases led to isolation of Psyp1 cDNA which is most abundantly expressed in short roots.

Psyp1 is the first peroxidase cDNA to be isolated from the genus Pinus. It encodes a 363‐amino‐acid class‐III peroxidase with a calculated molecular mass of 35.7 kDa and theoretical pI of 4.74. The predicted PSYP1 amino‐acid sequence is grouped with other class‐III peroxidases in phylogenetic analyses, but it has a unique amino‐acid sequence which may be associated with its function in short roots or with its phylogenetic group. The presence of a signal sequence for extracellular transport indicates that PSYP1 belongs to the group of secreted class‐III peroxidases. The presence of 10 tyrosine residues and putative auxin‐binding regions in PSYP1 suggests that the function of the enzyme is associated with cell‐wall formation in short roots. The downregulation of Psyp1 expression in symbiotic short roots hosting the ectomycorrhizal fungus Suillus bovinus is perhaps related to the change in cell‐wall structure necessary for ectomycorrhizal development.
Δ9‐Tetrahydrocannabinol increases nerve growth factor production by prostate PC‐3 cells
Authors: Velasco Lara , Ruiz Lidia , Sánchez Maria G. , Díaz‐Laviada Inés
Published: March 2003

Cannabinoids, the active components of marihuana, exert a variety of effects in humans. Many of these effects are mediated by binding to two types of cannabinoid receptor, CB1 and CB2. Although CB1 is located mainly in the central nervous system, it may also be found in peripheral tissues. Here, we study the effect of cannabinoids in the production of nerve growth factor by the prostate tumor cell line PC‐3. We show that addition of Δ9‐tetrahydrocannabinol to PC‐3 cells stimulated nerve growth factor production in a dose‐dependent and time‐dependent manner. Maximal effect was observed at 0.1 µmΔ9‐tetrahydrocannabinol and 72 h of treatment. Stimulation was reversed by the CB1 antagonists AM 251 and SR 1411716A. Pre‐treatment of cells with pertussis toxin also prevented the effect promoted by Δ9‐tetrahydrocannabinol. These results indicate that Δ9‐tetrahydrocannabinol stimulation of nerve growth factor production in these cells was mediated by the cannabinoid CB1 receptor. The implication of Raf‐1 activation in the mode of action of Δ9‐tetrahydrocannabinol is also suggested.
Mapping DNA‐binding sites of HIV‐1 integrase by protein footprinting
Authors: Dirac Annette M. G. , Kjems Jørgen
Published: March 2003

The HIV‐1 integrase protein catalyzes integration of the viral genome into host cell DNA. Whereas the structures of the three domains of integrase have been solved separately, both the structural organization of the full‐length protein and its interaction with DNA remain unresolved. A protein footprinting approach was employed to investigate the accessibility of residues in the full‐length soluble integrase mutant, INF185K,C280S, to proteolytic attack in the absence and presence of DNA. The N‐terminal and C‐terminal domains were relatively more accessible to proteolytic attack than the core domain. The susceptibility to proteolytic attack was specifically affected by DNA at residues Lys34, in the N‐terminal domain, Lys111, Lys136, Glu138, Lys156–Lys160, Lys185–Lys188, in the core domain, and Asp207, Lys 215, Glu246, Lys258 and Lys273 in the linker and C‐terminal domain, suggesting that these regions are involved in, or shielded by, DNA binding. Lys34 is positioned in a putative dimerization domain, consistent with the notion that DNA stabilizes the dimeric state of integrase.
Phosphatidylinositol 3‐kinases in tumor progression
Authors: Roymans Dirk , Slegers Herman
Published: March 2003

Many cellular processes have been identified in which phosphatidylinositol 3‐kinase has a key regulatory function. As an oncogene, it is also involved in the development of cancer. The transformation and progression of normal cells towards an advanced stage tumor and/or towards metastatic lesions involves a complex series of events, including genetic alterations, leading to aberrant cell cycle progression, altered adhesion and motility characteristics, inhibition of apoptosis and induction of angiogenesis. This review highlights the processes involved in the pathogenesis of cancer in which phosphatidylinositol 3‐kinase is involved and provides an overview of the possible mechanisms by which the enzyme exerts its oncogenic action.
Metallothionein isogene transcription in red blood cell precursors from human cord blood
Authors: Rahman Mohammad T , De Ley Marc
Published: March 2003

The in vitro transcription patterns for 10 functional metallothionein (MT) isogenes have been investigated in red blood cell (RBC) precursors from human cord blood. Active transcription status of the isogenes, MT‐0, MT‐1A, MT‐1B, MT‐1E, MT‐1G, MT‐1X, and MT‐2A, was detected in both ex vivo expanded RBC precursors (burst‐forming unit‐erythroid) and glycophorin A+ and CD71+ cells separated by magnetic cell sorting. Transcription patterns of these isogenes were analyzed at different times of incubation with the addition of Zn supplement. In neither the ex vivo expanded precursors nor glycophorin A+ and CD71+ cells could MT‐1F and MT‐3 be detected. Transcripts of MT‐4 were detected in glycophorin A+ and CD71+ cells. Erythropoietin‐responsive constitutive transcription of MT‐1X and possible interleukin‐3‐responsive downregulation of MT‐2A in ex vivo expanded precursors reveal their effect on MT biosynthesis. Biosynthesis and induction of MT at the protein level in the RBC precursors was also demonstrated by immunoblotting.
Hydrolysis of glycine‐containing elastin pentapeptides by LasA, a metalloelastase from Pseudomonas aeruginosa
Authors: Vessillier Sandrine , Delolme Frédéric , Bernillon Jacques , Saulnier Joëlle , Wallach Jean
Published: March 2003

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in vulnerable hosts. It may produce various virulence factors including proteases. Among them, LasA possesses both elastolytic and staphylolytic (hydrolysis of pentaglycine cross‐links in the cell wall peptidoglycan) activities. To understand if its elastolytic activity results from a preference for glycine‐rich substrates, we studied its ability to hydrolyse the 65 pentapeptides of human tropoelastin containing at least three glycines. As demonstrated by capillary electrophoresis (CE), 22 of these peptides were hydrolysed by LasA, generally at a single peptide bond and the catalytic ratio kcat/KM was determined for most of them. The highest value was obtained for LGGGA, 59 ± 9 min−1·mmol−1·L. The specificity of hydrolysis was elucidated by CE, liquid secondary ion mass spectrometry and, in some cases, collision activated dissociation‐mass analysis of ion kinetic energy. The preferred cleavage sites are GG and GA peptide bonds, the sequence GG↓A being especially sensitive to hydrolysis. Both positions P2 and P′2 must be occupied for hydrolysis and the presence of an amino acid in P3 (but not in P′3) significantly increases the catalytic ratio. Considering these results, about 30 GGX sequences (X: G, A or Y) of human tropoelastin could be susceptible to LasA elastolysis.
Organization and chromosomal localization of the murine Testisin gene encoding a serine protease temporally expressed during spermatogenesis
Authors: Scarman Anthony L. , Hooper John D. , Boucaut Kerry J. , Sit Mae‐Le , Webb Graham C. , Normyle John F. , Antalis Toni M.
Published: March 2003

The recently characterized human serine protease, Testisin, is expressed on premeiotic testicular germ cells and is a candidate type II tumor suppressor for testicular cancer. Here we report the cloning, characterization and expression of the gene encoding mouse Testisin, Prss21. The murine Testisin gene comprises six exons and five introns and spans ≈ 5 kb of genomic DNA with an almost identical structure to the human Testisin gene, PRSS21. The gene was localized to murine chromosome 17 A3.3‐B; a region syntenic with the location of PRSS21 on human chromosome 16p13.3. Northern blot analyses of RNA from a range of adult murine tissues demonstrated a 1.3 kb mRNA transcript present only in testis. The murine Testisin cDNA shares 65% identity with human Testisin cDNA and encodes a putative pre‐pro‐protein of 324 amino acids with 80% similarity to human Testisin. The predicted amino‐acid sequence includes an N‐terminal signal sequence of 27 amino acids, a 27 amino‐acid pro‐region, a 251 amino‐acid catalytic domain typical of a serine protease with trypsin‐like specificity, and a C‐terminal hydrophobic extension which is predicted to function as a membrane anchor. Immunostaining for murine Testisin in mouse testis demonstrated specific staining in the cytoplasm and on the plasma membrane of round and elongating spermatids. Examination of murine Testisin mRNA expression in developing sperm confirmed that the onset of murine Testisin mRNA expression occurred at ≈ day 18 after birth, corresponding to the appearance of spermatids in the testis, in contrast to the expression of human Testisin in spermatocytes. These data identify the murine ortholog to human Testisin and demonstrate that the murine Testisin gene is temporally regulated during murine spermatogenesis.
Diguanosine nucleotide metabolism and the survival of artemia embryos during years of continuous anoxia
Authors: Warner Alden H. , Clegg James S.
Published: March 2003

Encysted embryos of the primitive crustacean, Artemia franciscana, are remarkably resistant to a variety of harsh environmental conditions, including continuous anoxia for periods of years at physiological temperatures and water contents. Previous study produced no evidence of an ongoing anoxic metabolism, suggesting that these embryos remained viable in spite of the lack of detectable free energy flow and biosynthesis. That seeming violation of a major axiom of cell biology and biochemistry prompted us to re‐examine the nucleotide pool of encysted embryos during prolonged anoxia. We found that the nucleotide Gp4G, present initially in very large amounts, decreased slowly as anoxia continued over the 5.6‐year period examined. Studies on other nucleotides and associated enzymes, including results from previous papers, provide a plausible metabolic pathway leading to the provision of ATP and GTP to meet the needs of endergonic processes in anoxic embryos. Exactly what those processes are is not obvious. One possibility involves the extensive anoxia‐induced nuclear translocation of the stress protein, molecular chaperone p26, whose large molecular mass (≈ 500 kDa) most likely requires a supply of free energy to cross the nuclear envelope. Support for this possibility comes from our finding here that p26 is also a GTPase.
Expression of penicillin G acylase from the cloned pac gene of Escherichia coli ATCC11105
Authors: Dai Minghua , Zhu Yingmin , Yang Yunliu , Wang Enduo , Xie Yong , Zhao Guoping , Jiang Weihong
Published: March 2003

The structural gene pac in Eschericia coli ATCC11105 encodes penicillin G acylase (PGA). Within the pac gene, there is a regulatory gene pacR, which is transcribed in the opposite direction. Site‐directed mutagenesis was performed at base 1045 of pac by replacing a T with a C. This substitution did not alter the amino‐acid sequence of PGA, but changed the translation start codon of pacR from AUG to GUG. The expression of the mutant pacR decreased dramatically and the lacZ transcriptional fusion analysis showed that GUG was an extremely poor initiation codon for pacR. The pacR mutation caused PGA expression to be constitutive rather than inductive in two strains (E. coli A56, DH10B). The pac inducer phenylacetic acid (PAA) gave significant induction of PGA production at a concentration of 0.2% in wild type, but PAA at this concentration inhibited both cell growth and PGA production in the pacR mutated strains. The temperature‐dependent expression character of pac is preserved in the pacR translation‐initiation mutant and the optimum temperature of PGA production was 22 °C in both wild type and mutant. At a higher temperature of 37 °C, the PGA precursor polypeptide could not be matured into subunits and formed inclusion bodies, as revealed by western blot analysis. Our investigations confirmed the hypothesis of pacR‐mediated PAA induction for PGA expression and clarified the inhibitory effect of high temperature upon the post‐translational processing of the PGA precursor polypeptide.