bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of the 5′‐Flanking Regulatory Region of the Mouse Gene Encoding the Clearance Receptor for Atrial Natriuretic Peptide
Authors: Yanaka Noriyuki , Kotera Jun , Taguchi Ikuhiko , Sugiura Masaki , Kawashima Keisuke , Omori Kenji
Published: August 2004

A full‐length cDNA, encoding the mouse atrial natriuretic peptide clearance receptor (ANP‐CR), was isolated from a mouse lung cDNA library. The deduced amino acid sequence of the mouse ANP‐CR, showing a typical tripartite organization which lacks a guanylyl cyclase domain, was extremely well conserved compared with the ANP‐CR homologs. To understand the molecular mechanisms underlying the regulation of mouse ANP‐CR gene expression and to define the essential DNA sequences for the transcriptional activity, a genomic clone containing over 9 kb of the 5′‐flanking region of the mouse ANP‐CR gene has been isolated from a mouse genomic library. Sequence analysis revealed that the 2.3‐kb region upstream from an ATG codon of the mouse ANP‐CR gene contained a number of putative regulatory elements; TATA box, CAAT box, cAMP response element, AP‐1 and two shear stress responsive elements. Additionally, an unusual feature was the presence of the tandem‐repeated AP‐2‐like elements, which were closely overlapped with SP‐1 element. Promoter analysis using deletion plasmids in mouse Balb/3T3 cells, highly producing ANP‐CR mRNA, demonstrated that deletion of the sequence from −144 to +46 relative to the transcription start point caused a dramatic decrease of the transcriptional activity and that the TATA box at −269 was not essential for the basal transcriptional activity. Primer extension analysis indicated that transcription of the mouse ANP‐CR gene starts from at least two major sites, suggesting that the sequence from −144 to +46, which was shown to involve a novel sequence composed of tandem‐repeated TATA‐box‐like elements, contained promoter sequences. Furthermore, cis acting negative elements were shown to be situated in three regions (from −1178 to −708, from −707 to −625 and from −248 to −145) of the mouse ANP‐CR gene promoter.
31P Solid‐State NMR Measurements used to Detect Interactions between NADPH and Water and to Determine the Ionisation State of NADPH in a Protein‐Ligand Complex Subjected to Low‐Level Hydration
Authors: Gerothanassis Ioannis P. , Barrie Patrick J. , Birdsall Berry , Feeney James
Published: August 2004

31P‐NMR spectra of NADPH and NADPH bound to Lactobacillus casei dihydrofolate reductase have been recorded using the techniques of cross‐polarization, magic‐angle spinning and high‐power proton‐decoupling on both lyophilized and hydrated samples. Previous studies on the lyophilized complex of L. cusei dihydrofolate reductase with NADPH and methotrexate, measuring the isotropic shifts and principal components of the chemical shift tensors, have shown that the 2′‐phosphate group of bound NADPH exists as a mixture of the dianionic and monoanionic states [Gerothanassis, I. P., Barrie, P. J., Birdsall, B. & Feeney, J. (1994) Eur. J. Biochem. 226, 211–218]. In the present study on hydrated samples, the characterization of the isotropic shift and chemical shift tensors of the 2′‐phosphate signal indicates that the 2′‐phosphate is almost exclusively in the dianionic state. This is in agreement with earlier 31P‐NMR studies in solution [Feeney, J., Birdsall, B., Roberts, G. C. K. & Burgen, A. S. V. (1975) Nature 257, 564–566], In experiments examining progressively hydrated (6%, 12%, 15%, by mass) samples, the observed signals become increasingly narrower probably because the microenvironments of the 31P nuclei become more homogeneous upon sample hydration. Chemical exchange between mobile water molecules and bound protons close to individual sites on NADPH has been indirectly monitored on a hydrated sample (15% water, by mass) using a pulse sequence proposed by Harbison and coworkers [Harbison, G. S., Roberts, J. E., Herzfeld, J. & Griffin, R. G. (1988) J. Am. Chem. Soc. 110, 7221–7223]. In this experiment, the two diphosphate signals are totally suppressed while the 2′‐phosphate phosphorus signal remains: this indicates a significant polarization of the 2′‐phosphate nuclei from protons in exchange with those of mobile water molecules.
Formation of Pores in Escherichia coli Cell Membranes by a Cecropin Isolated from Hemolymph of Heliothis virescens Larvae
Authors: Lockey Timothy D. , Ourth Donald D.
Published: August 2004

The insect humoral defense system produces antibacterial peptides called cecropins. Cecropins were initially isolated from Hyalophora cecropia pupae and have since been isolated and identified in various insects. In this study, we have isolated and identified a cecropin from Heliothis virescens larvae. Rabbit IgG were raised against synthetic cecropin B. Affinity chromatography with the rabbit anti‐(cecropin B) IgG was used to isolate a cecropin from hemolymph of H. virescens larvae. Acid gel electrophoresis followed by a bacterial‐overlay analysis showed that Heliothis cecropin is a basic peptide of low molecular mass with bactericidal activity against Escherichia coli K12 D31. Heliothis cecropin is therefore analogous to synthetic cecropin B. One unresolved issue concerning cecropins and other antibiotic peptides is the mode of action by which they kill bacteria. By means of electron microscopy and immunocyto‐chemistry with gold‐labeled rabbit anti‐cecropin IgG, binding of purified and synthetic cecropin to the cell membranes of E. coli K12 D31 cells was observed. Small lesions in the cell membrane were seen that had a diameter of 9.6 nm and internal pore of 4.2 nm. The Heliothis cecropin was found to be a pore‐forming molecule that causes lesions in the cell membrane of E. coli K12 D31. The lesions lead to leakage of cytoplasmic contents and death of bacteria.
Microenvironmental Changes in Platelet Membranes Induced by the Interaction of Fibrinogen‐Derived Peptide Ligands with Platelet Integrins
Authors: Watała Cezary , Gwoździński Krzysztof , Pluskota Elżbieta , Dzieciątkowska Elżbieta , Cierniewski Czesław S.
Published: August 2004

A few studies have confirmed the influence of peptides containing either the RGD or dodecapeptide H‐12‐V (HHLGGAKQAGDV) sequence on cell membrane structure and function. In order to consider previous findings and to explore microenvironmental changes associated with the interaction of these two fibrinogen‐derived peptides with platelet membranes, we employed fluorescence quenching and electron paramagnetic resonance techniques to monitor the possible alterations in platelet membrane dynamics induced by RGDS and H‐12‐V. The interaction of RGDS with platelet membranes resulted in reduced values of the h+1/h0 parameter in both 5‐doxylstearic acid and 16‐doxylstearic acid spectra indicating a significant rigidification of the membrane lipid bilayer. Otherwise, the fibrinogen‐derived peptide that contained the γ chain C‐terminal sequence H‐12‐V had a fluidizing effect on the platelet membrane lipid bilayer. The labelling of platelet membranes with 1‐anilino‐8‐naphthalenesulphonate (ANS) enabled us to estimate the energy transfer efficiency and the apparent interchromophore distance between membrane protein tryptophan and ANS embedded into the membrane lipid bilayer. As RGDS interacts with platelet membrane this distance decreases, resulting in the relevant increase of energy transfer efficiency. The opposite alterations were recorded upon interaction of platelet membranes with H‐12‐V. Furthermore, a small shift towards longer wavelengths, which accompanies the spectra of ANS in control platelet membranes, vanishes during the interaction with the peptide H‐12‐V. This observation can be accounted for by a decrease in the polarity of the ANS environment, and may suggest an enhanced contact of the membrane tryptophan with phospholipid fatty acids. Thus, the data indicate that after the action of H‐12‐V on platelet membrane receptors, the membrane tryptophan residues become exposed to the external environment and the quenchable fraction of membrane tryptophan becomes smaller. The increase (a) in the relative rotational correlation time (τc) of 4‐(ethoxyfluorophosphinyloxy)‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl (ethoxyfluorophosphinyloxy‐TEMPO) and (b) in the hw/hs ratio in the spectra of 4‐maleimido‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl (maleimido‐TEMPO) indicate that under these conditions there is an effective immobilization of some domains located on the hydrated surface of membrane proteins and mobilization of those domains buried inside the membrane protein molecules. The interaction of RGDS with platelet membrane integrins resulted in contrary effects, as compared to H‐12‐V. In conclusion, our spectroscopic data indicate that these two fibrinogen‐derived peptides induce opposite effects in the dynamics of platelet membrane components.
Purification and Characterization of Mitochondrial Ribonuclease P from Aspergillus Nidulans
Authors: Lee Young Chul , Lee Byeong Jae , Hwang Deog Su , Kang Hyen Sam
Published: August 2004

Mitochondrial ribonuclease (RNase) P from Aspergillus nidulans was purified to near homogeneity using whole‐cell extract as the starting material. A 4400‐fold purification with a yield of 5.2% was achieved by ammonium sulfate fractionation, heat treatment, and five types of column chromatography, including tRNA–affinity column chromatography. This enzyme, which has a molecular mass of 232 kDa determined by glycerol gradient sedimentation analysis, appears to be composed of seven polypeptides and an RNA moiety. These seven polypeptides consistently copurified with the RNase P activity through two ion‐exchange chromatography columns and in a glycerol gradient. As judged by nuclease sensitivity, the enzyme requires an RNA component for its activity. The 3′‐end‐labeled RNAs that copurified with the enzyme displayed identical sequences but had variable lengths for the 5′ end, indicating that they originated from a common RNA molecule, the putative RNA component of RNase P. The purified enzyme cleaved mitochondrial precursor tRNAHis, resulting in an 8‐bp acceptor stem. This implies that the purified RNase P is a mitochondrial enzyme and that an additional guanylate residue (at position −1) of tRNAHis in A. nidulans mitochondria is generated by a mode that is analogous to the generation of their counterparts in prokaryotes and chloroplasts.
Overexpression, Solubization and Purification of Rat and Human Olfactory Receptors
Authors: Nekrasova Ena , Sosinskaya Adel , Natochin Michael , Lancet Doron , Gat Uri
Published: August 2004

The superfamily of olfactory receptor genes, whose products are thought to be activated by odorant gands, is critical for odor recognition. Two olfactory receptors, olp4 from rat and OR17–4 from human, were overexpressed in Sf9 insect cells. The presence of the proteins in cell membranes was monitored by immunoblotting with peptide‐specific polyclonal antibodies directed against the C‐terminal sequences of these receptors and with a mAb against an N‐terminal octapeptide epitope tag. A DNA sequence that codes for a His6 tag, which binds tightly to a Ni2+‐chelate‐affinity column, was incorporated into the N‐termini of both genes. The expressed olfactory receptors were found mainly in the cell‐membrane fraction. The proteins were difficult to solubize by many detergents and only lysophosphatidylchone was found to be both suitable for efficient solubization of the overexpressed olfactory receptors and compatible with the purification system used. After solubization, the olfactory receptors were purified to near homogeneity by affinity chromatography on nickel nitrilotriacetic acid resin and by cation‐exchange chroma‐tography. Electrophoresis of the purified proteins and visuazation with Coomassie Blue staining or by immunoblotting with specific antibodies, revealed bands of 32, 69 and 94 kDa, which were identified as the monomeric, dimeric and trimeric forms of the receptor proteins. The ogomeric forms were resistant to reduction and alkylation, and are therefore thought to be held together by non‐covalent hydrophobic interactions that are resistant to SDS. This finding is similar to previous observations for other guanine‐nucleotide‐binding‐regulatory‐protein‐coupled receptors. Reconstitution in phosphopid vesicles showed that the purified olfactory receptors insert specifically into the pid bilayer. This provides a means to study functional reconstitution with putative transduction components such as olfactory guanine‐nucleo‐tide‐binding‐regulatory protein.
The RNA Component of Mitochondrial Ribonuclease P from Aspergillus Nidulans
Authors: Lee Young Chul , Lee Byeong Jae , Kang Hyen Sam
Published: August 2004

Several RNA molecules that copurified with Aspergillus nidulans mitochondrial ribonuclease (RNase) P were identified [Lee, Y. C., Lee, B. J., Hwang, D. S. & Kang, H. S. (1996) Eur. J. Biochem. 235, 289–296], and their partial sequences were determined. Using an oligonucleotide probe, we cloned and mapped the gene encoding this putative RNA component of RNase P (RNase P‐RNA), situated between URFA3 (unidentified reading frame A3) and cobA (apocytochrome b) genes in the mitochondrial genome of A. nidulans. The gene is extremely (A+T)‐rich and contains two regions of sequence similarity conserved among the known mitochondrial RNase P‐RNAs and the eubacterial RNase P‐RNAs. The determination of 5′ and 3′ termini by primer extension and sequencing indicated that the length of the RNA transcript is 232 nucleotides. Northern‐blot analysis revealed that its only subcellular location was the mitochondria. Two RNase P‐RNA fragments of 110 nucleotides and 80 nucleotides, each containing one of the two conserved regions, could be recovered from the nuclease‐treated enzyme without significant loss of activity. The sizes of these fragments appeared to be the minimum lengths required for the vitro activity of the enzyme.
Mechanistic Studies on the 8‐Amino‐7‐Oxopelargonate Synthase, a Pyridoxal‐5′‐Phosphate‐Dependent Enzyme Involved in Biotin Biosynthesis
Authors: Ploux Olivier , Marquet Andrée
Published: August 2004

The reaction mechanism of 8‐amino‐7‐oxopelargonate (8‐amino‐7‐oxononoate) synthase from Bacillus sphaericus, an enzyme dependent on pyridoxal 5′‐phosphate (pyridoxal‐P), which catalyzes the condensation of l‐alanine with pimeloyl‐CoA, the second step of biotin biosynthesis, has been studied. To facilitate mechanistic studies, an improved over‐expression system in Escherichia coli, and a new continuous spectrophotometric assay for 8‐amino‐7‐oxopelargonate synthase were designed. In order to discriminate between the two plausible basic mechanisms that can be put forth for this enzyme, that is: (a) formation of the pyridoxal‐P ‐stabilized carbanion by abstraction of the C2‐H proton of the alanine‐pyridoxal‐P aldimine, followed by acylation and decarboxylation, and (b) formation of the carbanion by decarboxylation followed by acylation, the fate of the C2‐H proton of alanine during the course of the reaction has been examined using 1H NMR. Spectra of the 8‐amino‐7‐oxopelargonate formed using either l‐[2‐2H] alanine in H2O or l‐alanine in D2O, showed that the C2‐H proton of alanine is lost during the reaction and that the C8‐H proton of 8‐amino‐7‐oxopelargonate is derived from the solvent, a result that is only consistent with mechanism (a). Futhermore 8‐amino‐7‐oxopelargonate synthase catalyzes, in the absence of pimeloyl‐CoA, the stereospecific exchange, with retention of configuration, of the C2‐H proton of l‐alanine with the solvent protons. Similarly, 8‐amino‐7‐oxopelargonate synthase catalyzes the exchange of the C8‐H proton of 8‐amino‐7‐oxopelargonate. In addition to these exchange reactions, 8‐amino‐7‐oxopelargonate synthase catalyzes an abortive transamination yielding an inactive pyridoxamine 5′‐phosphate (pyridoxamine‐P) form of 8‐amino‐7‐oxopelargonate synthase and pyruvate. Kinetic analysis gave a rate constant of kexch= 1.8 min−1 for the exchange reaction which is 10 times lower than the catalytic constant and a rate constant of ktrans= 0.11 h−1 for the transamination. Finally deuterium kinetic isotope effects (KIE) were measured at position 2 of L‐alanine (DV= 1.3) and in D2O (D2OV= 4.0). The magnitudes of the KIE are consistent with a partially rate‐limiting abstraction of the C2‐H proton of alanine and a partially rate‐limiting reprotonation step. Taken together, all these results show that 8‐amino‐7‐oxopelargonate synthase utilizes mechanism (a). 8‐Amino‐7‐oxopelargonate synthase and 5 aminolevulinate synthase, which has also been shown to use mechanism (a), belong to a class of pyridoxal‐P ‐dependent enzymes that catalyze the formation of α‐oxoamines. Based on the fact that all these α‐oxoamine synthases share strong sequence similarities, we postulate that they also share the same reaction mechanism.
A Molecular Map of the Interactions between Titin and Myosin‐Binding Protein C
Authors: Freiburg Alexandra , Gautel Mathias
Published: August 2004

The thick filaments of vertebrate striated muscles contain with myosin a number of accessory proteins of the intracellular immunoglobulin superfamily, which are localized in a distinct pattern of stripes 43 nm apart. The specific localization of these proteins is believed to be due partly to their interaction with the giant muscle protein titin (also called connectin), which spans the entire sarcomere and may act as a molecular ruler. We have used recombinant fragments of titin covering the thick filament region to investigate their interaction with myosin‐binding protein C (MyBP‐C) from skeletal and cardiac muscle. The interaction of titin and MyBP‐C is directed by a subset of titin immunoglobulin domains that are specific for the C‐region of the thick filament, supporting the ruler hypothesis for the myosin‐binding proteins. The interaction of recombinant titin with overlapping fragments of human cardiac MyBP‐C maps the titin‐binding site within the C‐terminal region, which is deleted in patients suffering from the chromosome‐11–associated form of familial hypertrophic cardiomyopathy. This disorder is therefore likely to be the result of thick‐filament misassembly by abolishing the ternary interaction of titin, myosin and MyBP‐C.
Phytanic Acid is a Retinoid X Receptor Ligand
Authors: Lemotte Peter K. , Keidel Siegfried , Apfel Christian M.
Published: August 2004

Metabolic defects in phytanic acid catabolism have been shown to be connected with a number of human diseases which can lead to lethal defects of the nervous system and other organs. These effects are probably a result of the very high accumulation of phytanic acid in tissues throughout the body, due to defects in phytanic acid oxidation, the peroxisome being a major site for this process. The nuclear hormone receptors peroxisome proliferator‐activated receptor and retinoid X receptor (RXR) have been shown to function as transcription factors in the control of the peroxisomal enzyme expression. Known activators of peroxisome proliferator‐activated receptor include polyunsaturated fatty acids and, for RXR, the 9‐cis isomer of retinoic acid. Here we report that phytanic acid is also a natural ligand for RXRα, being able to activate a RXR‐responsive promoter. We present evidence that phytanic acid binds to RXRα, promotes formation of an RXRα/RXR response element complex (as detected by gel retardation), and induces a RXRα conformational change similar to that induced by 9‐cis‐retinoic acid (as detected by protease sensitivity). These results suggest an involvement of RXRα in the control of fatty acid metabolism and could imply that RXRs have a role in the disease effects resulting from defective phytanic acid catabolism.
Expression and Characterization of Recombinant α‐Galactosidase in Baculovirus‐Infected Insect Cells
Authors: Zhu Alex , Wang Zhong‐Kun
Published: August 2004

A cDNA encoding coffee bean α‐galactosidase was subcloned into baculovirus expression vectors, pVL‐1393 and pAc‐GP67B, for intracellular and extracellular expression in Spodopteru frugiperda (Sf9) insect cells, respectively. The expressed protein (recombinant α‐galactosidase) was immunologically reactive with antisera raised against its native counterpart isolated from coffee beans and was biologically active towards the substrate p ‐nitrophenyl α‐galactopyranoside. The subcellular distribution of recombinant α‐galactosidase expressed from different vectors was analyzed by Western blotting, immunofluorescent labeling, and electron microscopy. In addition, recombinant α‐galactosidase was compared to the native enzyme with respect to glycosylation, thermostability, and pH profile. Furthermore, a recombinant α‐galactosidase molecule with a His, tag at its C‐terminus was constructed by an overlap PCR method so that the enzyme expressed in Sf9 cells can be purified by a simple affinity chromatography procedure.
A Nonribosomal System of Peptide Biosynthesis
Authors: Kleinkauf Horst , Von Döhren Hans
Published: August 2004

This review covers peptide structures originating from the concerted action of enzyme systems without the direct participation of nucleic acids. Biosynthesis proceeds by formation of linear peptidyl intermediates which may be enzymatically modified as well as transformed into specific cyclic structures. The respective enzyme systems are constructed of biosynthetic modules integrated into multienzyme structures. Genetic and DNA‐sequence analysis of biosynthetic gene clusters have revealed extensive similarities between prokaryotic and eukaryotic systems, conserved principles of organisation, and a unique mechanism of transport of intermediates during elongation and modification steps involving 4′‐phospho‐pantetheine. These similarities permit the identification of peptide synthetases and related aminoacyl‐ligases and acyl‐ligases from sequence data. Similarities to other biosynthetic systems involved in the assembly of polyketide metabolites are discussed.
Association of Lyn Tyrosine Kinase with the Nuclear Matrix and Cell‐Cycle‐Dependent Changes in Matrix‐Associated Tyrosine Kinase Activity
Authors: Radha Vegesna , Nambirajan Sundaram , Swarup Ghanshyam
Published: August 2004

The nuclear matrix isolated from HeLa cells and Rat2 fibroblasts harbor? tyrosine kinase and tyrosine phosphatase activities. Polypeptides of 53, 56 and 60 kDa, associated with this subnuclear structure, were phosphorylated at tyrosine in vivo. By immunoblot and immunolabelling experiments, we identified one of the nuclear‐matrix‐associated tyrosine kinases as Lyn, a Src family member. Lyn was distributed as foci throughout the matrix. The p56 and p53 isoforms of Lyn remained firmly associated with the nuclear matrix after a variety of matrix preparation procedures, and were not detectable in the chromatin fraction of the nucleus. The tyrosine kinase activity associated with the nuclear matrix showed cell‐cycle‐dependent changes, maximum activity being observed at the G1/S transition phase. Polyoma‐virus‐transformed rat fibroblast cells showed sixfold higher tyrosine kinase activity in the nuclear matrix preparations compared to that in untransformed cells. These observations are consistent with the suggestion that tyrosine kinase activity associated with the nuclear matrix may be an important determinant of cellular proliferation.
NMR Structural Studies of DNA Decamer Duplex Containing the Dewar Photoproduct of Thymidylyl(3′→5′)Thymidine
Authors: Hwang Geum‐Sook , Kim Jong‐Ki , Choi Byong‐Seok
Published: August 2004

The single‐stranded deoxynucleotide decamer containing a site‐specific Dewar valence isomer of the (6–4) adduct of thymidylyl (3′→5′)‐thymidine was generated by direct photolysis of d(CGCATTACGC) with UV‐B and UV‐C irradiation. The conformation of the Dewar‐photomodified deoxyoligonucleotide duplex, (C1‐G2‐C3‐A4‐TS[DW]T6‐A7‐C8‐G9‐C10)·(G11‐C12‐G13‐T14‐A15‐A16‐T17‐G18‐C19‐G20), has been studied by one‐ and two‐dimensional NMR spectroscopy. While the eight of the ten complementary nucleotides form Watson‐Crick‐type hydrogen bonding, the 5′‐TpT‐3′ bases of the Dewar lesion show no evidence of complementary hydrogen bonding formation to each other. The Dewar covalent linkage for the adjacent pyrimidine base leads to unusual base stacking, which is different from that of normal B‐DNA. Unusual NOEs indicate that the formation of a Dewar photoproduct in the B‐DNA duplex is likely to alter its local and global structures. Also, detailed NMR data show that the base pairing and stacking of the Dewar‐photoproduct‐containing decamer duplex differ from that of the (6–4)‐adduct‐containing decamer duplex, suggesting that isomerization of the (6–4) adduct to its Dewar form induces a substantial change in the structure of the oligonucleotide duplex.
Alternative Splicing of the Pyruvate Kinase M Gene in a Minigene System
Authors: Takenaka Masaru , Yamada Kazuya , Lu Ting , Kang Rujun , Tanaka Takashi , Noguchi Tamio
Published: August 2004

The M2‐type and M2‐type isozymes of pyruvate kinase are produced from a single gene by mutually exclusive use of exons 9 and 10. Selection of exon 10 generates the M2type, which occurs in most tissues, whereas the M2type is expressed by use of exon 9 only in skeletal muscle, heart and brain. We investigated the mechanism by which exon 10, but not exon 9 is selected in M2‐expressing cells by transfecting minigenes containing exon 9 and/or exon 10 in cells and by analyzing the transcripts using reverse‐transcriptase polymerase chain reaction. Deletion of the most conserved region in intron 8 did not affect selection of exon 10 in dRLh‐84 cells, which express only the M2type. Exclusion of exon 10 from the minigene resulted in two major spliced products. One included correctly spliced exon 9 and the other skipped this exon. Similar splicing patterns were observed when these minigenes were transfected in hepatocytes which express the L type, but not M1or M2types. The 5′ splice site but not the 3′ splice site of exon 9 was found to be hardly recognized by the splicing machinery in dRLh‐84 cells. Mutation of the 5′ splice site sequence of exon 9 to that of exon 10 andvice versadid not change the splicing patterns. However, mutation of this site of exon 9 to a perfectly complementary sequence of U1 snRNA resulted in selection of exon 9 correctly spliced to exon 10. A 9–10 fusion exon (constructed by substitution of 68 bases of the 3′ portion of exon 9 and 33 bases of the 5′ portion of intron 9 for the corresponding regions of exon 10 and intron 10) was also correctly incorporated into a major product together with exon 10. Thus, we propose that exon 9 is not recognized in non M1‐expressing cells due to the weak signal of its 5′ splice site and that, although the 5′ splicing signal of exon 10 also appears to be weak, this exon can be recognized in these cells because the 5′ recognition signal may be relatively strengthened bycis‐acting element(s) which may be present in the 3′ portion of exon 9 and the 5′ portion of intron 9 and/or the corresponding regions of exon 10 and intron 10.
cDNA Cloning, Expression, and Chromosomal Localization of Caenorhabditis elegans DNA Topoisomerase I
Authors: Kim Jonghwa , Kim You‐Chan , Lee Jong Hyouk , Jang Yeon Joo , Chung In Kwon , Koo Hyeon‐Sook
Published: August 2004

By screening Caenorhabditis elegans cDNA libraries, overlapping cDNA clones encoding DNA topoisomerase I were obtained. An open reading frame of 751 amino acids was found in the 3.2‐kb cDNA sequence. The open reading frame has 54% and 50% identities to the amino acid sequences of human and Drosophila melanogaster DNA topoisomerases I, respectively. Northern blot analysis showed the presence of an mRNA of 3.4 kb which suggests that the cDNA sequence is close to full length. The 72‐kDa C‐terminal polypeptide expressed in Escherichia coli cells showed catalytic DNA topoisomerase I activity. The DNA topoisomerase I gene was mapped to position 18 of chromosome I by screening polytene YAC plasmid DNAs.
Human Biliverdin IXα Reductase is a Zinc‐Metalloprotein
Authors: Maines Mahin D. , Polevoda Bogdan V. , Huang Tian‐Jun , Mccoubrey William K.
Published: August 2004

Biliverdin TXα reductase (BVR) catalyzes the conversion of the heme b degradation product, biliverdin, to bilirubin. BVR is unique among enzymes characterized to date in that it has dual pH/cofactor (NADH, NADPH) specificity. A cDNA clone encoding human BVR was isolated from a λ library using a probe generated via reverse transcription and the polymerase chain reaction from human placental RNA. This approach was taken because the more direct approach of using the previously isolated rat BVR cDNA as the hybridization probe did not succeed. The human cDNA was cloned and sequenced; it was shown to have an open reading frame encoding a 296‐amino‐acid protein in which could be identified four peptides previously identified by micro‐sequencing purified protein. The cDNA hybridized with a single message of ≈ 1.2 kb in human kidney poly(A)‐rich RNA, and appeared, by Southern blot analysis, to be the product of a single‐copy gene. Sequence analysis indicated that the human reductase shows ≈83% identity, at both the nucleotide and amino acid levels, with rat BVR. In some regions including the carboxyl terminus, protein sequence identity drops to 45%. Also noteworthy is the presence of two additional cysteine residues in the encoded human reductase (five compared to three for rat). The protein produced by an expression plasmid in which the insert was cloned in frame with lac Z sequences was characterized, and demonstrated dual pH and cofactor dependence. However, as suggested by kinetic analysis, the human enzyme may also use NADH as cofactor, as opposed to the rat reductase, which most likely utilizes only NADPH under physiological conditions. Western blot analysis and iso‐electric focusing demonstrate that, although migrating as a single band on SDS/PAGE, the expressed protein, like that purified from tissue, consists of several isoelectric charge variants. Atomic absorption spectroscopy indicates that the protein purified from human liver contains Zn at an approximately 1:1 molar ratio. That human BVR is a Zn metalloprotein was further substantiated by 65Zn exchange analysis of both the purified and the fusion protein expressed in Escherichia coli. Exogenous Zn also inhibits NADPH‐dependent, but not NADH‐dependent, activity. Hence, the NADH and NADPH binding regions are differentiated by their ability to interact with Zn; Fe‐hematoporphyrin, however, inhibited both NADH‐ and NADPH‐dependent activity.
The Translational Cis ‐Regulatory Element of Mammalian Ribosomal Protein mRNAs is Recognized by the Plant Translational Apparatus
Authors: Shama Silvian , Meyuhas Oded
Published: August 2004

The translational efficiency of mammalian ribosomal protein mRNAs correlates with the growth status of the cells and its control is mediated through a 5′ terminal oligopyrimidine tract (5′ TOP) common to all these mRNAs. In the present study, we demonstrate that the plant translational apparatus, as represented by wheat‐germ extract, discriminates against mammalian mRNAs containing this motif to the same extent as do quiescent mammalian cells. Moreover, mutations in the 5′ TOP, which abolish the growth‐dependent translational control of the respective mRNAs in mammalian cells, render these mRNAs refractory to discrimination in the plant cell‐free system. This selective discrimination reflects neither the specific instability of 5′ TOP‐containing mRNAs during the incubation in vitro nor a lower competitive potential for the cap‐binding protein. The lower in vitro translational efficiency of these mRNAs is an inherent feature which is independent of whether they were derived from polysomes or messenger ribonucleoprotein particles of the transfected mammalian cells. The conservation of the discriminatory property of the translational apparatus between the animal and plant kingdoms is discussed from mechanistic and evolutionary points of view.
1H NMR of High‐Potential Iron‐Sulfur Protein from the Purple Non‐Sulfur Bacterium Rhodoferax fermentans
Authors: Ciurli Stefano , Cremonini Mauro Andrea , Kofod Pauli , Luchinat Claudio
Published: August 2004

Oxidized and reduced forms of high‐potential iron‐sulfur protein (HiPIP) from the purple non‐sulfur photosynthetic bacterium Rhodoferux fermentans have been characterized using 1H‐NMR spectroscopy. Pairwise and sequence‐specific assignments of hyperfine‐shifted 1H‐NMR signals to protons of cysteine residues bound to the [4Fe‐4S]3+/2+ cluster have been performed using one‐dimensional NOE and exchange spectroscopy experiments. 1H‐NMR hyperfine shifts and relaxation rates of cluster‐bound Cys β‐CH2 protons indicate that in the [4Fe‐4S]3+ cluster one iron ion can be formally described as Fe(III), while electron density corresponding to one electron is unevenly delocalized onto the remaining three iron ions. This delocalization is effected by means of two different electronic distributions interconverting rapidly on the NMR time scale. The mechanism of paramagnetic proton relaxation, studied by analyzing longitudinal relaxation rates of Cys β‐CH2 protons in HiPIPs from six different sources as a function of the Fe‐S‐Cβ‐Cα dihedral angle, indicate that the major contribution is due to a dipolar metal‐centered mechanism, with a non‐negligeable contribution from a ligand‐centered dipolar mechanism which involves the 3p orbital of the Cys sulfur atom. A semi‐quantitative tool for extracting structural information from relaxation time measurements is proposed.
Molecular Cloning of Two Different Mannose‐Binding Lectins from Tulip Bulbs
Authors: Van Damme Els J. M. , Briké Filip , Winter Harry C. , Van Leuven Fred , Goldstein Irwin J. , Peumans Willy J.
Published: August 2004

Two lectins were isolated from the bulbs of Tulipa cv. Apeldoom and their corresponding cDNA clones analyzed. The first, called TxLMII (second mannose‐binding Tulipa hybrid lectin), is a novel mannose‐binding tulip lectin. Based on its molecular structure, carbohydrate‐binding specificity and amino acid sequence, TxLMII belongs to the superfamily of mannose‐binding monocot lectins which are also found in representatives of the plant families Amaryllidaceae, Alliaceae, Orchidaceae and Araceae. Molecular cloning of the second lectin, called TxLCI (first Tulipa hybrid lectin with complex specificity), allowed determination unambiguously of the molecular structure of this previously described protein. In addition, evidence is presented that each TxLCI subunit possesses a mannose‐binding site and an N‐acetylgalactosamine‐binding site, which act independently of each other. Both binding sites are located in a separate domain of the lectin polypeptide. Since the first domain of TxLCT shows sequence similarity to TxLMII, it is suggested that their genes evolved from a common ancestor.
Alterations of Lipoprotein Fluidity by Non‐Esterified Fatty Acids Known to Affect Cholesteryl Ester Transfer Protein Activity
Authors: Foucher Christelle , Lagrost Laurent , Maupoil Véronique , Le Meste Martine , Rochette Luc , Gambert Philippe
Published: August 2004

The aim of the present study was to investigate the effect of saturated, monounsaturated and polyun‐saturated non‐esterified fatty acids (NEFA) on lipoprotein fluidity by using the electron spin resonance (ESR) method. The fluidity of the lipid phase of lipoproteins was evaluated by calculating from ESR spectra the S parameter of three different positional isomers of spin‐labeled stearic acid incorporated into the lipoprotein. In non‐enriched lipoproteins, S values were higher in high‐density lipoprotein 3 (HDL3) than in low‐density lipoprotein (LDL) indicating that the surface of HDL, was more ordered. Prior incubation of lipoprotein particles with NEFA significantly reduced S values, indicating an increased lipoprotein fluidity as compared with non‐supplemented homologous samples. In NEFA‐enriched lipoproteins, the modifications in fluidity were shown to be dependent on the structure of the NEFA acyl carbon chains. Medium‐chain fatty acids [lauric (12:0) and myristic (14:0) acids] appeared to be better fluidizing molecules as compared with both. shorter [octanoic (8:O) and decanoic (10:0) acids] and longer [palmitic (16:0) and stearic (18:0)acids] homologues. In addition, introducing at least one double bond in the acyl carbon chain significantly increased the ability of NEFA to reduce S as compared with saturated homologues. In both LDL and HDL3, the extent of the modifications of the molecular mobility at the lipoprotein surface was dependent on the final NEFA/lipoprotein ratio. In conclusion, these results suggest that the ability of NEFA to modulate the activity of the cholesteryl ester transfer protein might relate in part to alterations in fluidity at the lipoprotein surface.
Cooperation of Tyrosine Kinases P72syk and P53/56lyn Regulates Calcium Mobilization in Chicken B Cell Oxidant Stress Signaling
Authors: Qin Suofu , Inazu Tetsuya , Takata Minoru , Kurosaki Tomohiro , Homma Yoshimi , Yamamura Hirohei
Published: August 2004

A chicken B cell line DT40 and its syk‐negative or lyn‐negative mutants were used to investigate the roles of protein‐tyrosine kinases in oxidant stress signaling. The data presented here for wild‐type cells demonstrate that hydrogen peroxide stimulates p53/56lyn‐dependent tyrosine phosphorylation and activation of p72syk, and induces a rapid and prolonged elevation of intracellular calcium, which consists of calcium release from intracellular stores and influx from the extracellular space. Hydrogen‐peroxide‐triggered calcium mobilization was impaired in both syk ‐negative and lyn ‐negative cells, which was mainly due to the loss of calcium release from intracellular stores. Further studies indicated that inositol trisphosphate production was also abolished in both syk ‐negative and lyn ‐negative cells, which is consistent with the loss of calcium release. Taken together, these observations suggest that the defect of p72syk or p53/56lyn was responsible for the abnormality of calcium mobilization in both lyn ‐negative and syk ‐negative cells, and that both p72syk and p53/56lyn might regulate calcium mobilization through the phospha‐tidylinositol pathway in B cell oxidant stress signaling.
The Myeloid Differentiation Antigen CD14 is N‐ and O‐Glycosylated
Authors: Stelter Felix , Pfister Martin , Bernheiden Martin , Jack Robert S. , Bufler Philip , Engelmann Hartmut , Schütt Christine
Published: August 2004

The myeloid differentiation antigen CD14 acts as the major receptor for bacterial lipopolysaccharide (LPS). A soluble form of the protein (sCD14) is present in human serum which functions as a soluble LPS receptor. We have compared the isoform patterns of soluble CD14 derived from human serum and of the recombinant proteins produced by CHO cells transfected with either the wild‐type CD 14 gene or with a cDNA coding for a truncated protein which lacks the C‐terminal 21 amino acids [sCD14‐(1–335)‐peptide]. Using SDS/PAGE, two dominant isoforms (53 and 50 kDa) and two minor forms (46 and 43 kDa) can be detected in serum as well as in the supernatants of both transfectants. sCD14 is a glycopro‐tein which carries N‐ and O‐linked carbohydrates. The different isoforms of sCD14‐(1–335)‐peptide are due to differences in the content of N‐linked sugars. However after the removal of N‐ and O‐linked carbohydrates from serum‐ and CHO‐derived wild‐type proteins, two isoforms are still present. These results indicate that N‐linked glycosylation contributes to but does not fully explain the different forms of soluble CD14. We further examined whether the mutation of individual N‐linked glycosylation sites influences the expression of membrane‐bound and soluble CD 14 forms and the ability of the membrane‐bound molecule to bind LPS. As with the wild‐type proteins, the different isoforms of the soluble mutants are partially due to differences in N‐linked glycosylation. A truncated mutant which lacks the two N‐terminal glycosylation sites ([Aspl8, Asp132]CD14‐(1–335)peptide) does not give rise to multiple forms on SDS gels. Like CD14‐(1–335)‐peptide, this mutant is not expressed on the cell surface suggesting that a smaller isoform present in the wild‐type preparations results from proteolytic cleavage of the membrane‐bound molecule. N‐linked carbohydrates do not seem to be important for the binding of LPS to membrane‐bound CD14.
Mutations of Glu92 in Ferredoxin I from Spinach Leaves Produce Proteins Fully Functional in Electron Transfer but Less Efficient in Supporting NADP+ Photoreduction
Authors: Piubelli Luciano , Aliverti Alesaandro , Bellintani Francesca , Zanetti Giuliana
Published: August 2004

Ferredoxin I in spinach chloroplasts fulfils the role of distributing electrons of low redox potential produced by photosystem I to several metabolic routes, NADP+ reduction being the major output. To investigate the role of Glu92, which is conserved in the chloroplast‐type ferredoxins, mutations of this residue to either Gln, Ala or Lys were obtained through site‐directed mutagenesis. A Glu93Ala mutant was also designed. The four mutants of ferredoxin I were overproduced in Escherichia coli, purified and characterised. The different migration in nondenaturing gel electrophoresis of wild‐type and mutant proteins confirmed that the desired mutation was present in the expressed proteins. Spectral and physical properties of the mutants were similar to those of wild‐type ferredoxin; electron‐transfer properties were, however, quite different in the case of the mutants at position 92. Unexpectedly, these mutant ferredoxins were found to be twice as active as the wild‐type protein in supporting the NADPH–cytochrome c reductase reaction catalysed by ferredoxin–NADP+ reductase. However, interactions of the mutant ferredoxins with the isolated thylakoid membranes deprived of endogenous ferredoxin showed that the mutants were less capable of supporting NADP+ photoreduction than the wild‐type protein: both V and the apparent Km for reduced ferredoxin were influenced. On the other hand, the Kd values for the complex between oxidised ferredoxin and the reductase, measured at low ionic strength, were substantially changed only in the case of the Glu→Lys mutation. With this mutant the rate of cross‐linking between the two proteins induced by a carbodiimide was also decreased. It was found that the redox potentials of the iron‐sulfur cluster of the mutants were more positive by 73–93 mV than that of ferredoxin I [Aliverti, A., Hagen, W. R. & Zanetti, G. (1995) FEBS Lett. 368, 220–2241, Thus, the behavior of the ferredoxin mutants can be rationalised in terms of the effect of the side‐chain replacement on the electrochemical properties of the [2Fe‐2S] cluster and of an impairment in the interaction with the reductase under physiological conditions.
The Incorporation of Arachidonic Acid into Triacylglycerol in P388D1 Macrophage‐Like Cells
Authors: Balsinde Jesús , Dennis Edward A.
Published: August 2004

When P388D1 cells are incubated in media containing 10 μM [3H] aragudibuc acid (4, Ach), the tabel is rapidly incorporated into phospholipids and triacylglycerol. However, incorporation of [3H] 4, Ach into phospholipids clearly precedes incorporation into triacylglycerol, indicating that the phospholipid pool constitutes the primary metabolic fate of the 4 Ach via a remodelling pathway. In contrast, [3H]4, Ach is incorporated into triacylglycerol almost exclusively via de novo synthesis, as evidenced by studies using propranolol, a phosphatidate phosphohydrolase inhibitor. This compound induced a time‐dependent and concentration‐dependent increase in the levels of [3H] 4 Ach‐containing phosphatidate that is directly correlated with a dccreasc in the levels or [3H] 4, Ach‐containing triacylglycerol. In addition, no change in the levels of [3H]4, Ach‐containing diacylglycerol and monoacylglycerol were apparent along the time course of fatty acid incorporation into triacylglycerol. However, a sharp and transient accumulation of cell‐associated free [3H]4 Ach wits detected shortly after exposure of the cells to the radioactive fatty acid.

Collectively, the results reported herein suggest that free 4, Ach availability determines the patterns of incorporation and distribution of this fatty acid among the various lipid classes of P388D, cells.
CCAAT/Enhancer‐Binding Protein β (C/EBPβ) Binds and Activates While Hepatocyte Nuclear Factor‐4 (HNF‐4) does not Bind but Represses the Liver‐Type Arginase Promoter
Authors: Chowdhury Shoaib , Gotoh Tomomi , Mori Masataka , Takiguchi Masaki
Published: August 2004

In an attempt to elucidate the mechanism governing liver‐specific transcription of the arginase gene, we previously detected two protein‐binding sites designated footprint areas A and B at positions around –90 and –55 bp, respectively, relative to the transcription start site of the rat arginase gene. Based on the finding that area A was bound by a liver‐selective factor(s) related to CCAAT/enhancer‐binding protein (C/EBP), we performed cotransfection assay and showed that C/EBP family members and a related factor, albumin D‐element‐binding protein (DBP) stimulate transcription from the arginase promoter. In addition to area A, a recombinant C/EBPβ protein bound to area B, which appeared to be primarily responsible for activation by C/EBPs. We unexpectedly found that the arginase promoter activity stimulated by C/EBPs and DBP was repressed by another liver‐enriched transcription factor, hepatocyte nuclear factor‐4 (HNF‐4). Analysis of chimeras formed between the arginase promoter and the herpes simplex virus thymidine kinase promoter allowed us to delimit the negative HNF‐4‐responsive element into the region overlapping with footprint area B. However, no apparent binding of HNF‐4 was observed in this negative element. We speculate that HNF‐4 is involved in fine regulation of the arginase gene in the liver or shutdown of the gene in nonhepatic tissues without direct binding to the promoter region.
The Gene, Protein and Glycan Structures of Laccase from Pleurotus ostreatus
Authors: Giardina Paola , Aurilia Vincenzo , Cannio Raffaele , Marzullo Liberato , Amoresano Angela , Siciliano Rosa , Pucci Piero , Sannia Giovanni
Published: August 2004

A member of the laccase multigene family in Pleurotus ostreatus has been cloned and sequenced. The gene structure has been determined by comparison with the corresponding cDNA, synthesized by reverse transcription/PCR amplification.

The gene encode a laccase isoenzyme of 533 amino acids which has already been purified and characterized [Palmieri, G., Giardina, P., Marzullo, L., Desiderio, B., Nitti, G., Cannio, R. & Sannia, G. (1993) Appl. Microbiol. Biotechnol. 39, 632–636]. More than 92% of the protein sequence, including the N and C termini, has been verified by fast‐atom‐bombardment mass spectrometry, thus confirming the correspondence between the gene and its protein product.

The protein was N‐glycosylated at Asn444. Glycan analysis showed the presence of only a high‐mannose structure containing varying numbers of mannose residues. The presence of O‐linked oligosaccharides as well as other post‐translational modification could be ruled out by the miss analysis.
Oxygenic Photosynthesis
Authors: Nugent Jonathan H. A.
Published: August 2004

Photosystems I and II drive oxygenic photosynthesis. This requires biochemical systems with remarkable properties, allowing these membrane‐bound pigment‐protein complexes to oxidise water and produce NAD(P)H. The protein environment provides a scaffold in the membrane on which cofactors are placed at optimum distance and orientation, ensuring a rapid, efficient trapping and conversion of light energy. The polypeptide core also tunes the redox potentials of cofactors and provides for unidirectional progress of various reaction steps. The electron transfer pathways use a variety of inorganic and organic cofactors, including amino acids. This review sets out some of the current ideas and data on the cofactors and polypeptides of photosystems I and II.