bluuebunny/crossref_metadata_2025 huggingface dataset:
m7GpppG Cap Dependence for Efficient Translation of Drosophila 70‐kDa Heat‐Shock‐Protein (Hsp70) mRNA
Authors: Song Hyun‐Joo , Gallie Daniel R. , Duncan Roger F.
Published: July 2010

To investigate whether preferential translation of the heat‐shock mRNAs occurs via cap‐independent translation, the requirement for the m7GpppG cap structure for efficient translation of 70‐kDa heat‐shock‐protein (Hsp70) mRNA was quantified by in vitro translation and by in vivo translation following electro‐poration. Hsp70 mRNA was transcribed in vitro with and without a cap structure. Translation in the rabbit reticulocyte or wheat germ lysate was reduced about 70% when the cap was absent. For comparison, translation of uncapped encephalomyocarditis virus 5′‐untranslated‐region‐containing mRNA was equal to or greater than capped mRNA, whereas translation of several non‐heat‐shock mRNAs was reduced by 85–95% when capping was omitted. Cap‐dependent translational stimulation of Hsp70 is not due to increased stability, is not a kinetic effect, and requires the methylated GpppG. To confirm the in vitro analyses, capped and uncapped mRNA were introduced into Drosophila tissue culture cells by electroporation, followed by heat shock. Paralleling the in vitro results, uncapped Hsp70 mRNA translation was 70–80% reduced relative to the capped form. Complementary experiments in which eIF‐4 was inactivated in vitro using either m7GTP cap analogue or foot‐and‐mouth‐disease virus L protease expression likewise indicated that the cap‐dependent translation pathway is required for optimal Hsp mRNA translation. Since cellular Hsp70 mRNA translation during heat shock is very efficient, it is unlikely that translation via a cap‐independent pathway is the principal basis for preferential translation.
Cell Cycle, Differentiation and Tissue‐Independent Expression of Ribosomal Protein L37
Authors: Su Shiawhwa , Bird R. Curtis
Published: July 2010

A unique human cDNA (hG1.16) that encodes a mRNA of 450 nucleotides was isolated from a subtractive library derived from HeLa cells. The relative expression level ofhG1.16during different cell‐cycle phases was determined by Northern‐blot analysis of cells synchronized by double‐thymidine block and serum deprivation/refeeding.hG1.16was constitutively expressed during all phases of the cell cycle, including the quiescent phase when even most constitutively expressed genes experience some suppression of expression. The expression level ofhG1.16did not change during terminal differentiation of myoblasts to myotubes, during which cells become permanently post‐mitotic. Examination of other tissues revealed that the relative expression level ofhG1.16was constitutive in all embryonic mouse tissues examined, including brain, eye, heart, kidney, liver, lung and skeletal muscle. This was unusual in that expression was not down‐modulated during differentiation and did not vary appreciably between tissue types. Analysis by inter‐species Northern‐blot analysis revealed thathG1.16was highly conserved among all vertebrates studied (from fish to humans but not in insects). DNA sequence analysis ofhG1.16revealed a high level of similarity to rat ribosomal protein L37, identifyinghG1.16as a new member of this multigene family. The deduced amino acid sequence ofhG1.16was identical to rat ribosomal protein L37 that contained 97 amino acids, many of which are highly positively charged (15 arginine and 14 lysine residues with a predictedMrof 11065). hG1.16 protein has a single C2‐C2zinc‐finger‐like motif which is also present in rat ribosomal protein L37. Using primers designed from the sequence ofhG1.16, unique bovine and rat cDNAs were also isolated by 5′‐rapid‐amplification of cDNA ends. DNA sequences of bovine and rat G1.16, clones were 92.8% and 92.2% similar to human G1.16 while the deduced amino acid sequences derived from bovine and rat cDNAs each differed by a single amino acid from the sequence ofhG1.16and the published rat L37 sequence. Southern‐blot analysis revealed thathG1.16exists in multiple copies in human, rat and mouse genomes. These G1.16 clones encode unique human, rat and bovine members of the ribosomal protein L37 gene family, which are constitutively expressed even during transitions from quiescence to active cell proliferation or terminal differentiation, in all tissues and all vertebrates investigated.
Degradation of Proteasomes by Lysosomes in Rat Liver
Authors: Cuervo Ana María , Palmer Amparo , Rivett A. Jennifer , Knecht Erwin
Published: July 2010

Proteasomes are high‐molecular‐mass multisubunit complexes which are believed, either by themselves or as a part of the 26S proteinase complex, to play a central role in extralysosomal pathways of intracellular protein breakdown. We have addressed the degradation of proteasomes in rat liver, investigating the possible role of lysosomes. Affinity‐purified antibodies against rat liver proteasomes were used for immunoblot analysis of isolated lysosomes. Although proteasomes are not found in lysosomes from normally fed rats, they were found to accumulate in lysosomes of rats treated with leupeptin (an inhibitor of lysosomal proteases) and could also be detected in lysosomes isolated from livers of starved (24 h) rats. Proteinase‐K treatment of these fractions, as well as immunogold procedures, show that a proportion of the proteasomes are inside lysosomes. Comparison of the amount of proteasomes found in lysosomes by immunoblotting with their experimentally determined half life (8.3 days) is consistent with an important role of these organelles in the degradation of rat liver proteasomes. Nevertheless, these data do not exclude the possibility that some nonlysosomal degradation of proteasome components also occurs. Since proteasomes were localized in autophagic vacuoles, it is likely that they are taken up mainly by nonselective autophagy. However, using an in vitro system, it was found that, under conditions of starvation, proteasomes may also be taken up into lysosomes and degraded via the heat‐shock cognate protein of 73 kDa (hsc73)‐mediated transport.
Reversible Super‐Reduction of the Cubane [4Fe‐4S](3+;2+;1+) in the High‐Potential Iron‐Sulfur Protein Under Non‐Denaturing Conditions
Authors: Heering Hendrik A. , Bulsink Yvonne B. M. , Hagen Wilfred R. , Meyer Terrance E.
Published: July 2010

The reversible 2×1 e− reduction of the cubane cluster from oxidized to reduced to super‐reduced states ([4Fe‐4S]3+⇄[4Fe‐4S]2+⇄[4Fe‐4S]1+) was studied in high‐potential iron‐sulfur proteins (HiPIPs). Super‐reduction to the 1 + state was not observed in any of the seven HiPIPs tested during cyclic voltammetry (down to −0.95 V). However, equilibration at low potential (pH 7.5) of Rhodopila globiformis HiPIP yields a transient peak around −0.47 V due to the oxidation of super‐reduced HiPIP adsorbed at the electrode. The peak area depends on the equilibration potential according to a one‐electron Nernst curve with a half‐wave potential at −0.91 V. Reduction of R. globiformis HiPIP with titanium(III)citrate at pH 9.5 is very slow [pseudo‐first‐order half‐life of 23 min with a 100‐fold excess Ti(III)] but is reversible, and the EPR spectrum with g values of 2.04 and 1.92 is similar to that of reduced [4Fe‐4S]1+ ferredoxins. Chemical or electrochemical reoxidation of the super‐reduced form resulted in an EPR spectrum with g∥= 2.12 and g⊥= 2.03, i.e. identical to that of oxidized HiPIP. From the equilibrium concentration of super‐reduced HiPIP at a low concentration of Ti(III), a reduction potential of −0.64 V can be estimated. Super‐reduction of the large HiPIP (iso‐2) from Rhodospirillum salinarum is also possible with Ti(III) (gz= 2.05) but the super‐reduced state is unstable. No super‐reduction with Ti(III) was observed for the other HiPIPs. The difference between the electrochemically observed reduction potential and oxidation potential is explained by a fast and reversible conformational change upon super‐reduction. The rate of super‐reduction with Ti(III) is limited by the small amount (0.1%) of HiPIP in the 2+ state with the super‐reduced conformation.
Structural Influence of Calcium on the Heme Cavity of Cationic Peanut Peroxidase as Determined by 1H‐NMR Spectroscopy
Authors: Barber Kathryn R. , Rodríguez Marañón María José , Shaw Gary S. , Van Huystee Robert B.
Published: July 2010

The cationic isozyme of peanut peroxidase (CPRx) is one of many peroxidases which requires calcium for enzyme activity. It has been previously shown that it requires 2 mol calcium to coordinate to 1 mol CPRx, and its related peroxidases from the basidiomycete Phanerochaete chrysosporium (Lip) and isozyme C of horseradish (HRPc). X‐ray crystallographic studies of LiP have shown that calcium is ligated near the C‐terminus of helices proximal and distal to the heme, where it has been suggested to maintain the active site. To determine if such a mechanism was possible in CPRx, high resolution 1H‐NMR spectroscopy was used to study the effect of calcium on the environment of its heme group and the coordinating histidine residues. The low‐spin cyano complex of the enzyme (CPRxCN) was studied in order to assign the majority of the resonances arising from the protons in the heme pocket in both the presence and absence of bound calcium ions using two dimensional nuclear Overhauser effect spectroscopy (NOESY). The two calcium ions present in CPRxCN were removed by a non‐denaturing method and a calcium titration was performed and monitored by 1H‐NMR spectroscopy. These studies showed that the binding of both calcium ions in CPRx influenced the heme environment in a similar manner (Kd= 0.1 μM). In particular, calcium‐dependent changes in several heme resonances and the proximal and distal histidine residues suggest that calcium binding to CPRx causes some reorientation of these residues with respect to the active site.
Biosynthesis and Biological Activities of Lantibiotics with Unique Post‐Translational Modifications
Authors: Sahl Hans‐Georg , Jack Ralph W. , Bierbaum Gabriele
Published: July 2010

Lantibiotics are biologically active peptides which contain the thioether amino acid lanthionine as well as several other modified amino acids. They can be broadly divided into two groups on the basis of their structures: type‐A lantibiotics are elongated, amphiphilic peptides, while type‐B lantibiotics are compact and globular. In the last decade there has been a marked increase in research interest in these peptides due both to the novel biosynthetic mechanisms by which they are produced, as well as to their potential applications. Lantibiotics are synthesised on the ribosome as a prepeptide which undergoes several post‐translational modification events, including dehydration of specific hydroxyl amino acids to form dehydroamino acids, addition of neighbouring sulfhydryl groups to form thioethers and, in specific cases, other modifications such as introduction of d‐alanine residues from l‐serine, formation of lysinoalanine bridges, formation of novel N‐terminal blocking groups and oxidative decarboxylation of a C‐terminal cysteine. The genetic elements responsible for these specific modification reactions encode unique enzymes with hitherto unknown reaction mechanisms. Production of these peptides also requires accessory proteins including processing proteases, translocators of the ATP‐binding cassette transporter family, regulatory proteins and dedicated producer self‐protection mechanisms. While the principle biological activity of most type‐B lantibiotics appears to be directed at the inhibition of enzyme functions, the type‐A lantibiotics kill bacterial cells by forming pores in the cytoplasmic membrane.
Determination of Haem Electronic Structure in His‐Met Cytochromes c by 13C‐NMR
Authors: Turner David L.
Published: July 2010

The assignment of 13C resonances of nuclei α to the haem in horse ferricytochrome c is completed and the Fenni contact shifts are evaluated at 30°C and 50°C using empirical magnetic susceptibility tensors to correct for dipolar interactions. The Fermi contact shifts are fitted to a model of molecular orbitals of eg, symmetry, which are subject to a rhombic perturbation. A similar analysis is performed using published data for Pseudomonas aeruginosa cytochrome C551. The relationship between the orientation of the effective g tensor and that of the rhombic perturbation in these proteins is shown to agree with theoretical predictions. A comparison between the orientation of the rhombic perturbations and the crystal structures of horse cytochrome c and P. aeruginosa cytochrome c551, reveals that the orientation of the histidine and methionine axial ligands dominates the rhombic perturbation and that the two ligands have approximately equal influence. The magnitude of the perturbation shows that the orientation of the axial ligands has little effect on the haem redox potential. However, the relationship that is established between the magnetic susceptibility tensor, the partially filled haem molecular orbitals, and the orientation of the haem ligands offers a new source of precise structural information.
Duodenase, a New Serine Protease of Unusual Specificity from Bovine Duodenal Mucosa
Authors: Zamolodchikova Tatyana S. , Vorotyntseva Tatyana I. , Antonov Vladymir K.
Published: July 2010

In this paper, data are presented on purification and properties of a new serine endopeptidase (duodenase) isolated from bovine duodenum mucosa. The enzyme has been purified to homogeneity by combinations of ammonium sulphate fractionation, carboxymethyl‐cellulose 52 chromatography, and affinity chromatography on Sepharose 4B with Kunitz soybean trypsin inhibitor as a ligand. Some physicochemical properties of this protease have been investigated. The molecular mass of the purified duodenase was determined to be 29 ± 0.5 kDa by SDS/PAGE and G‐2000 SW column chromatography. The enzyme molecule is a single chain and the native enzyme is a monomeric protein. Its isoelectric point was estimated to be 10 ± 0.2. Duodenase has two forms (I and II) which possess similar properties but differ in their amino acid composition. The new protease is a glycoprotein and contains approximately 3.5% sugars.

The enzyme displays trypsin‐like and chymotrypsin‐like activities and hydrolyzes the amide bonds of substrates having Lys, Arg, Tyr, Phe and Leu residues at the P1 position. Duodenase is most active at pH 7.9–8.2.

Duodenase was irreversibly inhibited by diisopropylphosphofluoridate and phenylmethanesulphonyl fluoride, indicative of an activ‐site serine in this protease. α‐N‐Tosyl‐l‐lysine chloromethane and α‐N‐tosyl‐l‐phenylalanine chloromethane, which react with an active His, caused marked inhibition of trypsin‐like and chymotrypsin‐like activities of duodenase. The enzyme activity was strongly supressed by trypsin inhibitors from different sources (soybeans, bovine lungs and Lima beans). Chicken egg white ovomucoid had no effect on the duodenase activity.

The N‐terminal sequence of the native duodenase (24 amino acid residues) shows high similarity with those of human and murine cytotoxic T‐lymphocyte granzymes, human leukocyte cathepsin G and rat mast cell chymases.

The biological role of duodenase is discussed.
Association and Coexpression of Fatty‐Acid‐Binding Protein and Glycoprotein CD36 in the Bovine Mammary Gland
Authors: Spitsberg Vitaly L. , Matitashvili Elvina , Gorewit Ronald C.
Published: July 2010

The involvement of glycoprotein CD36 and fatty‐acid‐binding protein (FABP) in cellular growth, differentiation, lipid transport and metabolism led us to examine the possible biochemical and physiological relationship(s) between these two proteins. We investigated three aspects of this relationship. We first attempted to identify any physical complex formed between CD36 and FABP in bovine milk fat globule membranes. These membranes are the product of mammary gland secretory epithelial cells. The second aspect studied was the effect of synthetic peptide analogs to the C‐terminus (amino acid residues 121–131) of bovine mammary gland FABP on cell proliferation, as a result of the interaction of these peptides with the ectodomain of CD36. Finally, mammary gland CD36 and FABP coexpression was defined at different stages of lactation and during involution. Immunoprecipitation, Western immunoblotting with anti‐FABP and anti‐CD36, Northern‐blot analysis and a mammary epithelial cell proliferation assay demonstrated that: (a) bovine milk fat globule membranes contain the complex of CD36 and FABP, and that this complex is, most likely, formed as a result of FABP binding to the cytoplasmic segments of CD36; (b) synthetic analog of the C‐terminus of FABP with the sequence Val‐Thr‐Cys, identical to the sequence found in the CD36‐binding domain of thrombospondin, was a more potent inhibitor of bovine mammary gland epithelial cell proliferation than a synthetic peptide with the Val‐Cys‐Thr sequence; (c) the expression of FABP and CD36 is related to the state of mammary cell differentiation, since it reaches its maximum during lactation and declines during the involutionary period.
Duodenase, a New Serine Protease of Unusual Specificity from Bovine Duodenal Mucosa
Authors: Zamolodchikova Tatyana S. , Vorotyntseva Tatyana I. , Nazimov Igor V. , Grishina Galina A.
Published: July 2010

The complete amino acid sequence of duodenase, a new serine endopeptidase from bovine duodenal mucosa, has been determined. The sequence was reconstructed by the automated sequence analysis of the peptides obtained after cleavage with trypsin, Staphylococcus aureus V8 protease, cyanogen bromide and duodenase. The enzyme is composed of 226 amino acid residues yielding a molecular mass of 29.06 kDa. The presence of six cysteine residues and one potential sugar‐chain‐binding site at Asn50 was revealed. A predicted catalytic triade characteristic of the serine proteases was traced in the duodenase primary structure at the corresponding positions (His44, Asp87 and Ser181 in the sequence). Comparison of the sequence of duodenase with the other known primary structures of mammalian serine proteinases reveales the duodenase identity to granzymes from human and mice, human cathepsin G and mast cell chymases from rat, and gives an overall sequence identity of 47–55% with the mentioned enzymes. Alignment of the known serine protease and duodenase primary structures showed unique amino acid residues within the duodenase substrate‐binding pocket at positions 189 (Asn) and 226 (Asp) (the bovine chymotrypsinogen A numbering). These results are discussed with respect to the relation between the duodenase unique residues within the primary specificity pocket S1, and the unusual dual specificity of the enzyme.
The Saccharide Chain of Lupin Seed Conglutin γ is not Responsible for the Protection of the Native Protein from Degradation by Trypsin, but Facilitates the Refolding of the Acid‐Treated Protein to the Resistant Conformation
Authors: Duranti Marcello , Gius Cristina , Sessa Fabio , Vecchio Giuseppe
Published: July 2010

Native glycosylated and enzymically deglycosylated conglutin γ (a lupin seed oligomeric protein) both showed an unusual resistance to tryptic degradation. The result of this treatment was that a single 40–residue peptide was cleaved from the N‐terminus of conglutin γ light subunit. Acid treatment of the two protein forms led to their substantial unfolding, as indicated by CD spectra. After this treatment, both polypeptides were completely degraded by trypsin after a few minutes of incubation. Conversely, trypsin pulse experiments run under renaturing conditions demonstrated a different refolding behaviour of the two proteins: the glycosylated form became resistant to trypsin after a 7‐h renaturation, while the deglycosylated form required 42 h renaturation. These results were confirmed by CD spectra and reverse‐phase HPLC analyses of the glycosylated and deglycosylated conglutin γ forms. Therefore, it was concluded that the saccharide chain of conglutin γ increased the rate of formation of a trypsin‐resistant conformation upon refolding of the acid‐treated protein, without playing any direct role in the protection of the native protein from proteolysis.
Formylmethanofuran: Tetrahydromethanopterin Formyltransferase (Ftr) from the Hyperthermophilic Methanopyrus kandleri
Authors: Shima Seigo , Weiss David S. , Thauer Rudolf K.
Published: July 2010

Methanopyrus kandleri is a methanogenic Archaeon that grows on H2 and CO2 at a temperature optimum of 98 °C. The gene ftr encoding the formylmethanofuran: tetrahydromethanopterin formyl‐transferase, an enzyme involved in CO2 reduction to methane, has been cloned, sequenced, and overexpressed in Escherichia coli. The overproduced enzyme could be purified in yields above 90% by simply heating the cell extract to 90°C in 1.5 M K2HPO4 pH 8.0 for 30 min. From 1 g wet cells (70 mg protein) approximately 14 mg formyltransferase was obtained. The purified enzyme showed essentially the same catalytic properties as that purified from M. kandleri cells. The primary structure and properties of the formyltransferase are compared with those of the enzyme from Methanobacterium thermoautotrophicum (growth temperature optimum 65 °C) and Methanothermus fervidus (83 °C). Of the three enzymes that from M. kandleri had the lowest isoelectric point (4.2) and the lowest hydrophobicity of the amino acid composition. The enzyme from M. kandleri had the relatively highest content in alanine, glutamate and glutamine and the relatively lowest content in isoleucine, leucine and lysine. These properties, some of which are unusual for enzymes from other hyperthermophilic organisms, may reflect that the formyltransferase from M. kandleri is adapted to both hyperthermophilic and halophilic conditions.
Inhibition of CYP2E1 Activity does not Abolish Pulsatile Urine Alcohol Concentrations During Chronic Alcohol Infusions
Authors: Badger Thomas M. , Ronis Martin J. J. , Ingelman‐Sundberg Magnus , Hakkak Reza
Published: July 2010

Continuous, long‐term intragastric infusions of ethanol leads to a two‐step induction of hepatic cytochromeP450 2E1 (CYP2E1) that is correlated with blood ethanol concentrations (BECs) and urine alkohol concentrations (UECs). In addition, long‐term and continuous ethanol infusion does not produce a steady‐state BEC, but results in pulsatile BECs and UECs having peak‐peak duration of approximately 6 days and ethanol concentrations ranging from near zero to over 500 mg/dl. In the present study, rats were treated with ethanol (levels reaching 13 g · kg−1· day−1) for 38 days in the presence of compounds reported to block CYP2E1 activity or expression, to study the possible involvement of CYP2E1 in the pulsatile BECs. The inhibitors used were chlormethiazole (CMZ); diallysulfide (DAS); phenethyl isothiocyanate (PET) and dihydrocapsacin (DHC). Hepatic microsomal metabolism of carbon tetrachloride andp‐nitrophenol, as well as mean CYP2E1 apoprotein levels, were significantly greater (P≤0.05) in ethanol‐treated rats than in control rats, whereas rats treated with DAS, CMZ or PET had significantly (P≤0.05) reducedp‐nitrophenol and carbon tetrachloride metabolism and lower CYP2E1 apoprotein levels compared to those of ethanol controls. UECs were variable in all ethanol‐treated groups and there was a typical pulsatile pattern that had a mean interpulse interval (the number of days between the peaks of two consecutive pulses) ranging over 5.4±0.3–6.0±0.7 days and a mean amplitude (nadir to peak UEC) of 415 ± 39–337 ± 33 mg/dl. None of the putative CYP2E1 blockers altered the pulsatile nature of ethanol in urine. Our results suggest that pulsatile UECs are not the result of variations in the amount of CYP2E1.
Differences in the DNA‐Binding Properties of the Hmg‐Box Domains of HMG1 and the Sex‐Determining Factor SRY
Authors: Teo Soo‐Hwang , Grasser Klaus D. , Thomas Jean O.
Published: July 2010

High‐mobility‐group protein 1 (HMG1) is an abundant, non‐sequence‐specific, chromosomal protein with two homologous, HMG‐box, DNA‐binding domains, A and B, and an acidic tail. The HMG‐box motif also occurs, as a single copy, in some sequence‐specific transcription factors, e.g. the sex‐determining factor, SRY. We have investigated whether or not there are differences in the DNA‐binding properties of the isolated A and B HMG‐box domains of HMG1 and SRY and whether, in the case of A and B, there might also be differences due to different sequence contexts within the native protein. The basic regions that flank the HMG1 B box, giving B', enhance its DNA‐binding, supercoiling and DNA‐bending activities, and promote the self‐association of the DNA‐bound B‐box. All the HMG‐box domains bind with structure specificity to four‐way junctions, but the structure selectivity is significantly greater for A and the SRY box than for the HMG1 B or B' domains, as judged by competition with excess plasmid DNA. The domains self‐associate to different extents on supercoiled DNA and this may explain differences in the ability to discriminate between four‐way junctions and supercoiled DNA. The HMG1 A, B and B' domains constrain negative superhelical turns in DNA, but the SRY HMG box does not. Only the full B domain (B') bends DNA in a ligase‐mediated circularisation assay; the minimal B box, the A domain and the SRY box do not. Thus, despite a common global fold, the HMG box appears to have been adapted to various functions in different protein contexts.
O‐Glycosylation of the Thr70 Residue of Cell‐Adhesive Lysozyme in Yeast
Authors: Yamada Takao , Uyeda Atsuko , Takao Toshifumi , Shimonishi Yasutsugu , Matsushima Masaaki , Kikuchi Masakazu
Published: July 2010

The cell‐adhesive protein Cys‐RGD4 has been constructed using a yeast expression system by inserting the sequence Cys‐Arg‐Gly‐Asp‐Ser‐Cys (CRGDSC) between Val74 and Asn75 of human lysozyme [Yamada, T., Uyeda, A., Kidera, A. & Kikuchi, M. (1994b) Biochemistry 33, 11678–11683]. The Cys74a, Arg74b, Gly74c, Asp74d, Ser74e, Cys74f‐lysozyme mutant, purified from the yeast culture supernatant contained glycosylated variants, in addition to the unglycosylated form. Peptide mapping analyses suggested that the glycosylation occurred at the Thr70 residue in the Cys‐RGD4 molecule. Electrospray ionization mass spectrometric analysis demonstrated the presence of two hexose residues in the major variant, and one, three, four, or five hexose residues in the minor variants. All of these hexose residues were identified as mannose by analysis of the oligosaccharide mixture obtained by mild alkaline treatment of the variants. No other glycosylation was observed, although the Cys‐RGD4 molecule possesses a total of 12 threonine and serine residues. In addition, the Thr70 residue is not glycosylated in either native lysozyme or the Arg‐Gly‐Asp‐Ser (RGDS)‐inserted mutant, RGD4 [Yamada, T., Matsushima, M., Inaka, K., Ohkubo, T., Uyeda, A., Maeda, T., Titani, K., Sekiguchi, K. & Kikuchi, M. (1993) J. Biol. Chem. 268, 10588–10592], Thus, this O‐glycosylation seems to be specific for both the mutant lysozyme molecule and the site of the threonine residue. Structural analyses of these lysozymes by X‐ray crystallography suggest that the conformation of the serine‐containing or threonine‐containing region can affect the specificity of yeast O‐glycosylation.
Isolation and Characterization of Mutants Affected in Lipid Metabolism of Chlamydomonas Reinhardtii
Authors: Sato Norihiro , Tsuzuki Mikio , Matsuda Yoshihiro , Ehara Tomoko , Osafune Tetsuaki , Kawaguchi Akihiko
Published: July 2010

Two mutants affected in lipid metabolism of Chlamydomonas reinhardtii were isolated by treating cells with ultraviolet light. Both mutants showed high chlorophyll fluorescent yields, as compared with parents, and were designated as hf‐2 and hf‐9 (for high fluorescence), hf‐2 was shown to be defective in the synthesis of a chloroplast‐specific lipid, sulfoquinovosyl diacylglycerol. hf‐9 was shown to be defective in desaturation at the ω6 position of fatty acids of monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol and phosphatidylglycerol. The mutants exhibited alterations in photosynthetic activity and chloroplast ultrastructure.
Structures of the N‐Linked Oligosaccharides on Human Plasma Vitronectin
Authors: Ogawa Haruko , Yoneda Atsuko , Seno Nobuko , Hayashi Masao , Ishizuka Ineo , Hase Sumihiro , Matsumoto Isamu
Published: July 2010

The structures of N‐linked oligosaccharides present on human plasma vitronectin were elucidated. Oligosaccharides were released from the vitronectin by N‐glycosidase F digestion and tagged with 2‐aminopyridine; the pyridylamino‐oligosaccharides were then fractionated by anion‐exchange and reverse‐phase HPLC, Ten major pyridylamino‐oligosaccharides were isolated. The linkages and locations of sialic acid residues were determined by desialylation with Salmonella sialidase in combination with acid. The asialo forms were then analyzed by two‐dimensional sugar mapping, component sugar analysis and 400‐MHz 1H‐NMR spectroscopy. The major oligosaccharides of human vitronectin were of the diantennary N‐acetyllactosamine type, with a lesser amount of the tri‐ and a small amount of the mono‐antennary type, to which 1–3 mol sialic acid residues were linked, mostly through α2‐6 linkages, although α2‐3 linkages were also present. The possibility that several binding activities of vitronectin can be ascribed to its glycan moiety was discussed, based on the specific features of the N‐linked oligosaccharides on human vitronectin revealed here.
Characterization of a Human Perinatal Myosin Heavy‐Chain Transcript
Authors: Jullian Eric H. , Kelly Alan M. , Pompidou Alain J. , Hoffman Rebecca , Schiaffino Stefano , Stedman Hansell H. , Rubinstein Neil A.
Published: July 2010

Using a monoclonal antibody specific to the neonatal myosin heavy chain, we have cloned the full‐length heavy chain cDNA from an 18‐week human fetal cDNA library. Ribonuclease protection assays were used to survey a human muscle collection ranging from 11 weeks gestation to 16 years. Expression of the RNA encoded by this cDNA was observed at 20 and 21 weeks gestation and at 2 days after birth. No expression was observed at 13.5 weeks, before 2 years, at 2 years, or after 2 years gestation. Due to the timing of its expression, this cDNA appears to represent of the human fetal myosin heavy chain. Sequencing of the entire 6010 bases showed high similarity to the rat perinatal myosin heavy chain [Periasamy, M., Wieczorek, D. F. & Nadal‐Ginard, B. (1984) J. Biol. Chem. 21, 13573–13578]. However, moderate divergence was observed when compared to a previously described human perinatal myosin heavy chain [Karsch‐Mizrachi, I., Feghali, R., Shows, T. B. & Leinwand, L. A. (1990) Gene 89, 289–294; Feghali, R. & Leinwand, L. A. (1989) J. Cell Biol. 108, 1791–1797]. Restriction fragment‐length polymorphism analyses of sites in both the S1 and rod domains showed the presence of this fetal myosin heavy chain sequence in all 27 genomic samples examined. Restriction fragment‐length polymorphism analysis failed to find the previously described perinatal isoform in any sample.
Rotational Dynamics of Calcium‐Free Calmodulin Studied by 15N‐NMR Relaxation Measurements
Authors: Tjandra Nico , Kuboniwa Hitoshi , Ren Hao , Bax Ad
Published: July 2010

The backbone motions of calcium‐free Xenopus calmodulin have been characterized by measurements of the 15N longitudinal relaxation times (T1) at 51 and 61 MHz, and by conducting transverse relaxation (T2), spin‐locked transverse relaxation (T1ρ), and 15N‐{1H} heteronuclear NOE measurements at 61 MHz 15N frequency. Although backbone amide hydrogen exchange experiments indicate that the N‐terminal domain is more stable than calmodulin's C‐terminal half, slowly exchanging backbone amide protons are found in all eight α‐helices and in three of the four short β‐strands. This confirms that the calcium‐free form consists of stable secondary structure and does not adopt a ‘molten globule’ type of structure. However, the C‐terminal domain of calmodulin is subject to conformational exchange on a time scale of about 350 μs, which affects many of the C‐terminal domain residues. This results in significant shortening of the 15N T2 values relative to T1ρ, whereas the T1ρ and T2 values are of similar magnitude in the N‐terminal half of the protein. A model in which the motion of the protein is assumed to be isotropic suggests a rotational correlation time for the protein of about 8 ns but quantitatively does not agree with the magnetic field dependence of the T1 values and does not explain the different T2 values found for different α‐helices in the N‐terminal domain. These latter parameters are compatible with a flexible dumbbell model in which each of calmodulin's two domains freely diffuse in a cone with a semi‐angle of about 30° and a time constant of about 3 ns, whereas the overall rotation of the protein occurs on a much slower time scale of about 12 ns. The difference in the transverse relaxation rates observed between the amides in helices C and D suggests that the change in interhelical angle upon calcium binding is less than predicted by Herzberg et al. Strynadka and James [Strynadka, N. C. J. & James, M. N. G. (1988) Proteins Struct. Funct. Genet. 3, 1–17].
Analysis of the Iron‐Sulfur Clusters Within the Complex I (NADH: Ubiquinone Oxidoreductase) Isolated from Potato Tuber Mitochondria
Authors: Lin Tse‐I , Sled Vladimir D. , Ohnishi Tomoko , Brennicke Axel , Grohmann Lutz
Published: July 2010

The mitochondrial complex I (NADH: ubiquinone oxidoreductase) isolated from potato (Solanum tuberosum) has been investigated for the presence of iron‐sulfur clusters. EPR spectroscopic analysis detected signals arising from clusters N1, N2, N3 and N4. Quantitation of the content of iron and sulfur within the isolated complex I showed the preparation to contain 22.6 mol acid‐labile sulfide and 30.4 mol iron/mol complex I. The iron‐sulfur cluster composition of the plant complex I appears to be similar to the well‐known composition found in Neurospora crassa.
The PR55 and PR65 Subunits of Protein Phosphatase 2A from Xenopus laevis
Authors: Bosch Mariette , Cayla Xavier , Van Hoof Christine , Hemmings Brian A. , Ozon René , Merlevede Wilfried , Goris Jozef
Published: July 2010

cDNA clones encoding the 65‐kDa (PR65) and 55‐kDa (PR55) regulatory subunits of protein phosphatase 2A from Xenopus laevis were isolated by homology screening with the corresponding human cDNAs, and used to analyze the developmental expression patterns of these genes. The PR65 subunit was found to be encoded by two genes, termed XPR65α and XPR65β. The open reading frames of the α and β cDNAs both span 1767 bp, and predict proteins of 64.4 kDa and 65.3 kDa, respectively, that are 87% identical. The predicted amino acid sequence of XPR65α showed 95% and 84% identity with human PR65α and PR65β proteins, respectively, whereas the identity of XPR65β with the same proteins was 87% and 86.5%, respectively. Only one type of Xenopus PR55 (XPR55) was isolated that showed 93% and 84% similarity to human PR55α and PR55β, respectively. Analysis of the N‐terminal region of XPR55 with the same regions of human PR55α and PR55β, indicates that the XPR55 is the Xenopus homolog of the human PR55α isoform. Despite the overall similarity with PR55 from other species, XPR55 has an N‐terminal extention of at least 24 amino acids. In the ovary, a transcript of 2.8 kb, encoding the XPR65β, was predominantly expressed and these XPR65β mRNAs are present at a constant level during oogenesis until late embryogenesis. Expression of the 2.4–kb XPR65α was low until the larval stage, then dramatically increased. In all adult tissues except ovary, the 2.4–kb α‐specific mRNA was more abundant than the 2.8–kb β transcript. Two transcripts of 2.4 kb and 2.5 kb, encoding the XPR55 subunit, were detected at a constant level throughout Xenopus oogenesis and during embryogenesis. Both transcripts were also expressed at similar levels in all adult tissues, but in a tissue‐specific manner. Analysis of the XPR55 and XPR65 proteins using antibodies to recombinant proteins revealed that the overall levels of the two proteins were constant, in good agreement with mRNA data.
Site‐Directed Mutagenesis and Structure/Function Studies of Casein Kinase II Correlate Stimulation of Activity by the β Subunit with Changes in Conformation and ATP/GTP Utilization
Authors: Jakobi Rolf , Traugh Jolinda A.
Published: July 2010

Casein kinase II exists in vivo as an active holoenzyme consisting of catalytic α and/or α' and regulatory β subunits, which form a tetrameric structure of α2β2. Unlike most other protein kinases, casein kinase II uses both ATP and GTP effectively as phosphate donors. Two residues unique to the catalytic subunit of casein kinase II, Va166 and Trp176, were mutated to Ala66 and Phe176, respectively, the amino acids present in more than 95 % of the identified protein kinase sequences. Using recombinant α subunit expressed in Escherichia coli, the mutations have been previously shown to reduce the utilization of GTP by changing Km values for ATP and GTP and to reduce the approximate fivefold stimulation observed upon addition of the regulatory subunit [Jakobi, R. & Traugh, J. A. (1992) J. Biol. Chem. 267, 23894–23902]. In this study, the mutations are shown to affect the catalytic activity of the reconstituted holoenzyme by changing both Km and Vmax values. The Vmax for ATP is reduced by the mutation of Trp176 to phenylalanine, but no change is observed with GTP. The Va166 to alanine and Va166/Trp176 to alanine/phenylalanine mutations reduce the Vmax values for ATP and GTP to levels comparable to those of the catalytic subunits alone, indicating that changes in the stimulation of activity by the β subunit are due to changes in Vmax. Structural studies using ultraviolet CD spectroscopy show that changes in stimulation of Vmax by the β subunit are correlated with changes in the secondary structure; the extent of these changes is reduced by both mutations. Correlation of changes in secondary structure and stimulation of activity by the β subunit indicate that the formation of the wild‐type holoenzyme causes conformational changes in the active site, leading to an increased rate of reaction. As shown by the mutations, Va166 and Trp176 are involved both in the conformational changes and in the selectivity of ATP and GTP.
The Role of the Intralysosomal pH in the Control of Autophagic Proteolytic Flux in Rat Hepatocytes
Authors: Luiken Joost J. F. P. , Aerts Johannes M. F. G. , Meijer Alfred J.
Published: July 2010

Current methods to estimate changes in intralysosomal pH in hepatocytes do not discriminate between lysosomes and other intracellular acidic compartments. To obtain selective information on the change in lysosomal function in response to a change in lysosomal pH we have used the liberation of fluorescent 4‐methoxy‐2‐naphthylamide from low concentrations of lysyl‐alanyl‐4‐methoxy‐2‐naphthylamide, B substrate of lysosomal dipeptidylpeptidase II. Using permeabilized and intact hepatocytes, the activity of this enzyme in response to manipulations meant to increase the intralysosomal pH was compared with intralysosomal protein degradation and with the accumulation of [14C]chloroquine as a pH indicator of intracellular acidic compartments. The data show that changes in intralysosomal pH are indicated by changes in dipeptidylpeptidase II activity and that these are mainly due to a pH‐dependent change in substrate accumulation in the lysosomes.

Subsequently, the method was applied to establishing the extent to which an increase in intralysosomal pH can contribute to the inhibition of autophagic proteolysis in intact hepatocytes caused by a decrease in intracellular ATP, by an increase in amino acid concentration and by hypo‐osmotic cell swelling. The following observations were made. (a) Moderate changes in intracellular ATP do not affect the lysosomal pH. (b) Hypo‐osmotic cell swelling, which promotes inhibition of proteolysis by amino acids in freshly isolated hepatocytes, does not affect the lysosomal pH. (c) In addition to their known inhibitory effect on autophagic sequestration, amino acids (leucine in particular) can increase the lysosomal pH and thus inhibit intralysosomal protein degradation directly. (d) Low concentrations of the acidotropic agent methylamine increase the lysosomal pH without having an effect on autophagic proteolytic flux.

It is concluded that autophagic proteolysis is not controlled by changes in the lysosomal pH.
Relationship between hexokinase and cytokinin in the regulation of leaf senescence and seed germination
Authors: Swartzberg D. , Hanael R. , Granot D.
Published: July 2010

Arabidopsis hexokinase (AtHXK1), an enzyme that catalyses hexose phosphorylation, accelerates leaf senescence, whereas the plant hormone cytokinin inhibits senescence. Previous work in our laboratory has shown that isopentenyl transferase (IPT), a key gene in the biosynthesis of cytokinin, expressed under promoters of the senescence‐associated genes SAG12 or SAG13 (PSAG12::IPT and PSAG13::IPT, respectively), inhibits leaf senescence in tomato plants. To study the relationship between hexokinase and cytokinin in the regulation of leaf senescence, we created and analysed double‐transgenic tomato plants expressing both AtHXK1 and either PSAG12::IPT or PSAG13::IPT. We found that expression of IPT in the double‐transgenic plants could not prevent the accelerated senescence induced by over‐expression of AtHXK1. Since cytokinin inhibits senescence via an apoplastic invertase that produces extracellular hexoses, whereas AtHXK1 is an intracellular mitochondria‐associated hexokinase, our results suggest that intracellular sugar sensing via AtHXK1 is dominant over extracellular sugar sensing with regard to leaf senescence. Interestingly, the heterologous SAG12 and SAG13 promoters are also expressed in germinating tomato seed, around the radicle penetration zone, suggesting that seed germination involves a senescence process that is probably necessary for radicle emergence. Indeed, seed expressing PSAG12::IPT and PSAG13::IPT exhibited delayed radicle emergence, possibly due to delayed endosperm senescence.
Regeneration potency of mouse limbs
Authors: Masaki Hideki , Ide Hiroyuki
Published: July 2010

Mammalians have a low potency for limb regeneration compared to that of amphibians. One explanation for the low potency is the deficiency of cells for regenerating amputated limbs in mammals. Amphibians can form a blastema with dedifferentiated cells, but mammals have few such cells. In this paper, we report limb formation, especially bone/cartilage formation in amputated limbs, because bone/cartilage formation is a basic step in limb pattern regeneration. After the amputation of limbs of a neonatal mouse, hypertrophy of the stump bone was observed at the amputation site, which was preceded by cell proliferation and cartilage formation. However, no new elements of bone/cartilage were formed. Thus, we grafted limb buds of mouse embryo into amputated limbs of neonatal mice. When the intact limb bud of a transgenic green fluorescent protein (GFP) mouse was grafted to the limb stump after amputation at the digit joint level, the grafted limb bud grew and differentiated into bone, cartilage and soft tissues, and it formed a segmented pattern that was constituted by bone and cartilage. The skeletal pattern was more complicated when limb buds at advanced stages were used. To examine if the grafted limb bud autonomously develops a limb or interacts with stump tissue to form a limb, the limb bud was dissociated into single cells and reaggregated before grafting. The reaggregated limb bud cells formed similar digit‐like bone/cartilage structures. The reaggregated grafts also formed segmented cartilage. When the reaggregates of bone marrow mesenchymal cells were grafted into the stump, these cells formed cartilage, as do limb bud cells. Finally, to examine the potency of new bone formation in the stump tissue without exogenously supplied cells, we grafted gelatin gel containing BMP‐7. BMP induced formation of several new bone elements, which was preceded by cartilage formation. The results suggest that the environmental tissues of the stump allow the formation of cartilage and bone at least partially, and that limb formation will be possible by supplying competent cells endogenously or exogenously in the future.
Studies on the role of Dlx5 in regulation of chondrocyte differentiation during endochondral ossification in the developing mouse limb
Authors: Chin Hsian‐Jean , Fisher Melanie C. , Li Yingcui , Ferrari Deborah , Wang Chi‐Kuang Leo , Lichtler Alexander C. , Dealy Caroline N. , Kosher Robert A.
Published: July 2010

The homeodomain transcription factor Dlx5 has been implicated in the regulation of chondrocyte and osteoblast differentiation during endochondral ossification in the developing limb. In a gain‐of‐function approach to directly investigate the role of Dlx5 in chondrocyte maturation, we have used cartilage‐specific Col2a1‐Dlx5 promoter/enhancer constructs to target overexpression of Dlx5 to the differentiating cartilage models of the limbs of transgenic mice. Targeted overexpression of Dlx5 in cartilage rudiments results in the formation of shortened skeletal elements containing excessive numbers of hypertrophic chondrocytes and expanded domains of expression of Ihh and type X collagen, molecular markers of hypertrophic maturation. This suggests that hypertrophic differentiation is enhanced in response to Dlx5 misexpression. Skeletal elements overexpressing Dlx5 also exhibit a marked reduction in the zone of proliferation, indicating that overexpression of Dlx5 reduces chondrocyte proliferation concomitant with promoting hypertrophic maturation. Taken together these results indicate that Dlx5 is a positive regulator of chondrocyte maturation during endochondral ossification, and suggest that it regulates the process at least in part by promoting the conversion of immature proliferating chondrocytes into hypertrophying chondrocytes; a critical step in the maturation process.
The Epigenetics of Germ‐line Immortality: Lessons from an Elegant Model System
Authors: Furuhashi Hirofumi , Kelly William G.
Published: July 2010

Epigenetic mechanisms are thought to help regulate the unique transcription program that is established in germ cell development. During the germline cycle of many organisms, the epigenome undergoes waves of extensive resetting events, while a part of epigenetic modification remains faithful to specific loci. Little is known about the mechanisms underlying these events, how loci are selected for, or avoid, reprogramming, or even why these events are required. In particular, although the significance of genomic imprinting phenomena involving DNA methylation in mammals is now well accepted, the role of histone modification as a transgenerational epigenetic mechanism has been the subject of debate. Such epigenetic mechanisms may help regulate transcription programs and/or the pluripotent status conferred on germ cells, and contribute to germ line continuity across generations. Recent studies provide new evidence for heritability of histone modifications through germ line cells and its potential effects on transcription regulation both in the soma and germ line of subsequent generations. Unraveling transgenerational epigenetic mechanisms involving highly conserved histone modifications in elegant model systems will accelerate the generation of new paradigms and inspire research in a wide variety of fields, including basic developmental studies and clinical stem cell research.