bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of Action of Sarcoplasmic Reticulum Calcium‐Uptake Activators
Authors: Berrebi‐Bertrand Isabelle , Lahouratate Philippe , Lahouratate Véronique , Camelin Jean Claude , Guibert Jacqueline , Bril Antoine
Published: July 2004

The Ca2+ uptake by the sarcoplasmic reticulum (SR) can be affected by direct modulation of the Ca2+ pump or by removing the inhibitory effect of dephosphorylated phospholamban. The effect of these mechanisms was assessed using ellagic acid and 1‐(3,4‐dimethoxyphenyl)‐3‐dodecanone. Both compounds (30 μmol/l) enhanced SR‐Ca2+ uptake in rabbit cardiomyocytes by 65.3±413% and 44.3±56.7% for 1‐(3,4‐dimethoxyphenyl)‐3‐dodecanone and ellagic acid, respectively (at pCa 6.2). A similar effect was observed in cardiac SR microsomes (59.5±7.4% and 45.1±6.7) with 30 μmol/l 1‐(3,4‐dimethodoxyphenyl)‐3‐dodecanone and ellagic acid, respectively. 1‐(3,4‐DimethoxyphenyI)‐3‐dodecanone increased Ca2+ storage by cardiac SR microsomes mainly at high [Ca2+] with a 57% increase of Vmax, whereas ellagic acid increased Vmax to a smaller extent (22%) and stimulated Ca2+ uptake at lower [Ca2+] with a leftward‐shift of the pCa/ATPase relationship by pCa 0.24. Ellagic acid also differed from 1‐(3,4‐dimethoxylphenyl)‐3‐dodecanone in that it produced a Ca2+ sensitizing effect only in cardiac SR microsomes (by pCa 0.3) whereas 1‐(3,4‐dimethoxyphenyl)‐3‐dodecanone stimulated the ATPase, at saturating Ca2+, in both cardiac and skeletal muscle SR vesicles. It is suggested that 1‐(3.4‐dimethoxypheny1)‐3‐dodecanone stimulates directly the Ca2+‐ATPase activity, in contrast to ellagic acid which enhances the cardiac SR‐Ca2+ uptake by interacting with phospholamban, as confirmed by the lack of additive effect between ellagic acid and monoclonal antibodies raised against phospholamban. 1‐(3,4‐dimethoxyphenyl)‐3‐dodecanone and ellagic acid constitute attractive pharmacological tools to investigate the functional consequences of enhancing SR Ca2+, uptake by affecting different mechanisms.
In Vitro Mutagenesis of PH‐20 Hyaluronidase from Human Sperm
Authors: Arming Sigrid , Strobl Birgit , Wechselberger Christian , Kreil Gunther
Published: July 2004

The cDNA encoding PH‐20 hyaluronidase from human sperm has been mutated at five positions by in vitro mutagenesis. We have changed three acidic amino acids and two arginine residues that are conserved in the sequence of mammalian PH‐20 polypeptides as well as in the hyaluronidases from bee and hornet venom. Of the former, the mutants [Gln113]PH‐20 and [Gln249]PH‐20 had no detectable enzymatic activity; the mutant [AsnllllPH‐20 had about 3% activity. The mutant [Thr252]PH‐20 was also inactive, while [Gly176]PH‐20 had only about 1% activity. This indicates that the PH‐20 hyaluronidases, like numerous enzymes that hydrolyze glycosidic bonds, have acidic amino acids in their active site. Moreover, for the binding of the substrate, the polyanion hyaluronan, arginine residues appear to be essential.
Subunit c from the Sodium‐Ion‐Translocating F1F0‐ATPase of Propionigenium Modestum
Authors: Matthey Ulrich , Kaim Georg , Dimroth Peter
Published: July 2004

Escherichia coli strain PEF42 produces a sodium‐ion‐dependent hybrid F1F0‐ATPase consisting of the Propionigenium modestum subunits a, b, c and δ, of a hybrid a subunit and of the E. coli subunits β, γ and E. The gene encoding subunit c of the P. modestum F1F0‐ATPase was cloned into the pT7‐7 expression vector to yield plasmid pT7c. E. coli PEF42 was transformed with plasmid pT7c together with plasmid pGP1–2, which harbours the gene for the T7 RNA polymerase. The production of the P. modestum subunit c was induced by a temperature shift from 30°C to 42°C for 30 min and led to an increased concentration of this protein in the membrane of the host strain. The c subunit produced in E. coli moved as a monomer in dodecylsulfate electrophoresis. The protein was extracted from the cells with chlorofodmethanol, purified and incorporated into sodium dodecylsulfate micelles. Circular dichroism of subunit c in sodium dodecylsulfate showed a temperature‐stable spectrum (between 20–60°C) with a high proportion of a‐helical structure. Upon incubation of subunit c with [14C]dicyclohexylcarbodiimide the protein became labelled in a sodium‐ion‐dependent manner, similar to the labelling observed if the purified F1F0‐ATPase of P. modestum, was treated with the radioactive carbodiimide. The Na+‐specific site was therefore retained in the isolated c subunit dissolved in dodecylsulfate.
The Ribosomal S16 Protein of Escherichia Coli Displaying a DNA‐Nicking Activity Binds to Cruciform DNA
Authors: Bonnefoy Eliette
Published: July 2004

We have recently shown that the ribosomal S16 protein of Escherichia coli is a magnesium‐dependent DNase which introduces nicks into supercoiled DNA molecules [Oberto, J., Bonnefoy, E., Mouray, E., Pellegrini, O., Wikstrom, P. M. & Rouvière‐Yaniv, J. (1996) Mol. Microbiol. 19, 1319–13301. In this work we analysed the DNA‐binding and DNA‐nicking properties of S16 using two different approaches. Gel‐retardation assays showed that S16 is a structure‐specific DNA‐binding protein displaying a preferential binding for cruciform DNA structures. This specific binding to cruciform DNA was further investigated using a supercoiled plasmid carrying the origin of replication of E. coli (oriC) which is an (A+T)‐rich DNA region with abundant palindromic sequences susceptible of forming cruciform‐like structures in vivo. We show that the nicks introduced by S16 in oriC are not randomly positioned but are precisely localised near such palindromic sequences. In addition, the nicking activity of S16 appeared to be sequence dependent since the cuts introduced by S16 occurred next to an adenine, in most cases an unpaired adenine, usually followed by a GTT sequence. Overall these experiments indicate that S16 requires a cruciform‐like DNA structure to bind DNA and the presence of a particular sequence in order to introduce specific single‐stranded cuts into a DNA molecule.
Catalytic Characteristics of Tryparedoxin
Authors: Gommel Daniel U. , Nogoceke Everson , Morr Michael , Kiess Michael , Kalisz Henryk M. , Flohé Leopold
Published: July 2004

Tryparedoxin, a thioredoxin‐related protein from Crithidia fasciculata with a molecular mass of 16 kDa catalyses the reduction of a peroxiredoxin‐type peroxidase, Cf21, at the expense of trypanothione [Nogoceke, E., Gommel, D. U., Kieζ, M., Kalisz, H. M. & Flohé, L. E. (1997) Biol. Chem. Hoppe‐Seyler 378, 827–8361, The kinetic analysis of tryparedoxin revealed an enzyme substitution mechanism. The corresponding molecular event was elucidated to be a reversible oxidoreduction of the disulfide bridge in the thioredoxin‐related motif WCPPC. The amino‐proximal cysteine residue of this active site was more reactive in S‐alkylation experiments than the distal residue. The natural substrates of tryparedoxin, trypanothione and Cf21, could only be substituted by glutathione and glutathione disulfide with considerable loss in activity. The pronounced specificity of tryparedoxin is further accentuated by low limiting Km values for Cf21 and trypanothione (2.2 μM and 130 μM, respectively, as compared to 990 μM for gluthathione disulfide and an infinite value for glutathione). Tryparedoxin can therefore be classified as a trypanothione: peroxiredoxin oxidoreductase. The reduction of tryparedoxin by trypanothione appears to be the rate‐limiting step in the trypanothione‐dependent hydroperoxide reduction because(a) the regeneration of reduced tryparedoxin from the tryparedoxin‐trypanothione complex is rate limiting (kcal, 392 min−1), (b) the physiological trypanothione concentrations may not always saturate tryparedoxin, and (c) the rate constants for the net forward reaction of Cf21 are faster than those of the tryparedoxin reaction. The functional characteristics of tryparedoxin explain the limited capacity of trypanosomatids in coping with oxidative stress and qualify the enzyme as a potential target for the design of specific trypanocidal compounds.
A Conformational Study of the Human and Rat Encephalitogenic Myelin Oligodendrocyte Glycoprotein Peptides 35–55
Authors: Albouz‐Abo Samia , Wilson Jennifer C. , Bernard Claude C. A. , Von Itzstein Mark
Published: July 2004

Myelin oligodendrocyte glycoprotein (MOG), is considered an important central‐nervous system‐specific target autoantigen for primary demyelination in autoimmune diseases like multiple sclerosis. We have recently demonstrated that MOG or its derived peptide, MOG‐(35–55)‐peptide, are able to produce in animals, clinicopathologic signs that mimic multiple sclerosis. The rat MOG sequence spanning amino acids 35–55 [rMOG‐(35–55)‐peptide] differs from the human sequence [hMOG‐(35–55)‐peptide] by a single amino acid substitution, i.e. Pro42→Ser. Mice injected with rMOG‐(35–55)‐peptide snowed severe inflammation and demyelination throughout the central nervous system but, interestingly, mice injected with hMOG‐(35–55)‐peptide showed only a few foci of mild inflammation with no demyelination. Circular dichroism and nuclear magnetic resonance spectroscopy have been used to structurally characterise the bioactive peptides hMOG‐(35–55)‐peptide and rMOG‐(35–55)‐peptide. In 0.1 M K2HPO4/KOH, 90% H2O/D2O solutions, these derived peptides have been shown, by NMR spectroscopy, to adopt detectable levels of short‐range structure in equilibrium with unfolded conformers. On addition of 2,2,2‐trifluoro‐(2H3)ethanol, rMOG‐(35–55)‐peptide and hMOG‐(35–55)‐peptide adopt folded structures which have nuclear Overhauser enhancements characteristic of a poorly defined α‐helix over residues 44–51. There are some indications of secondary structure also evident in the N‐terminal region of rMOG‐(35–55)‐peptide. CD spectroscopy has revealed that in aqueous solution both peptides are unfolded but in 2,2,2‐trifluoroethanol and, at micellar concentrations of sodium dodecyl sulfate, rMOG‐(35–55)‐peptide and, to a lesser extent, hMOG‐(35–55)‐peptide adopt helical conformations. In contrast, at non‐micellar concentrations of SDS rMOG‐(35–55)‐peptide and hMOG‐(35–55)‐peptide adopt, according to CD spectroscopy, a β‐structure indicating that the peptides change conformation depending on the micro‐environment of the amino acids.
Nuclear Factor Y Controls the Basal Transcription Activity of the Mouse Platelet‐Derived‐Growth‐Factor β‐Receptor Gene
Authors: Ishisaki Akira , Murayama Takanori , Ballagi Andrea E. , Funa Keiko
Published: July 2004

To determine the regulatory mechanism of the expression of the mouse platelet‐derived growth factor (PDGF) β‐receptor gene, a 1.9‐kb 5′ flanking genomic fragment was cloned and analyzed. Site‐directed mutagenesis of a CCAAT motif, located 60 bp upstream of the transcriptional‐start site, completely abolished the promoter activity [Ballagi, A. E., Ishisaki, A., Nelin, J.‐O. & Funa, K. (1995)Biochem. Biophys. Res. Commun. 210, 165–175]. The sequence around the intact CCAAT motif was protected by in vitro DNase‐I‐footprinting analysis. Electrophoresis‐mobility‐shift assays with anti‐[nuclear factor Y(NF‐Y)]Ig revealed binding of the NF‐Y complex to the CCAAT box. Furthermore, the double‐stranded oligonucleotides corresponding to the sequence around the CCAAT motif were conjugated with DNA‐affinity magnetic beads. The binding proteins were affinity purified and identified as the NF‐Y transcription factor by western blotting. Our results indicate that NF‐Y controls the basal transcription activity of the mouse PDGF β‐receptor gene.
Stimulation of Sky Tyrosine Phosphorylation by Bovine Protein S
Authors: Nyberg Petra , He Xuhua , Härdig Ylva , Dahlbäck Björn , García De Frutos Pablo
Published: July 2004

Protein S is an anticoagulant vitamin‐K‐dependent plasma glycoprotein, which acts as a cofactor to activated protein C in the degradation of coagulation factors Va and VIIIa. It has been proposed that protein S has an additional function as a growth factor. Protein S and a structurally similar protein, Gas6, have been found to stimulate members of the Axl/Sky family of receptor tyrosine kinases. Human Gas6 is able to activate Axl and Sky. In contrast, while bovine protein S activates human Sky and its murine homologue, human protein S activates murine Sky but not the human receptor. In the present investigation, we studied the structural background of this species difference. Using protein S chimeras with domains from human and bovine origin, we found that only those chimeras with the steroid‐hormone‐binding globulin‐like (SHBG) region from bovine protein S activate human Sky, indicating that the SHBG region is essential for the interaction. This observation was confirmed by inhibition of Sky phosphorylation by C4b‐binding protein, a plasma protein that interacts tightly with the SHBG region of protein S. Another chimeric molecule, composed of the N‐terminal 4‐carboxyglutamic‐acid‐containing domain (Gla domain) and the two epidermal‐growth‐factor‐like domains of human factor IX, and the SHBG region of bovine protein S, stimulated the receptor less efficiently. Antibodies directed against the Gla domain of protein S, inhibited the activation of human Sky by bovine protein S. These results indicate that the N‐terminal domains of protein S are not essential for activation of the receptor, but contribute to the affinity of the interaction. Our data suggest that protein S might be a ligand of Sky in some species despite the lack of activity of human protein S on human Sky. The bovine/human protein S species difference will be a useful model to establish the structural requirements for the interaction between Sky and its ligands.
Characterisation of the Pterin Molybdenum Cofactor in Dimethylsulfoxide Reductase of Rhodobacter Capsulatus
Authors: Solomon Peter S. , Lane Ian , Hanson Graeme R. , Mcewan Alastair G.
Published: July 2004

Analysis of dimethylsulfoxide reductase from Rhodobacter capsulatus showed that it contained 1 mol Mo and 2 mol GMP. This indicates that the molybdenum cofactor in dimethylsulfoxide reductase is bis(molybdopterin guanine dinucleotide) molybdenum. The absorption spectrum of the molybdopterin guanine dinucleotide released from dimethylsulfoxide reductase after denaturation of the holoenzyme was compared with those of pterin standards of known redox state. The spectra were most similar to pterin standards in the dihydro state and oxidised state. The reduction of 2,6‐dichloroindophenol by molybdopterin guanine dinucleotide released from dimethylsulfoxide reductase and by pterin standards was also measured and approximately 2 mol electrons/2 mol molybdopterin guanine dinucleotide were found to reduce 2,6‐dichloroindophenol. These results are consistent with the presence of one molybdopterin guanine dinucleotide moiety with a pyrazine ring at the oxidation level of a dihydropteridine and one molybdopterin guanine dinucleotide moiety with a pyrazine ring at the oxidation level of a fully aromatic pteridine. It is suggested that the pyrazine ring of Q‐molybdopterin guanine dinucleotide is fully aromatic and contains a 5,6 double bond.
The Glycosylated Matrix Protein of Borna Disease Virus is a Tetrameric Membrane‐Bound Viral Component Essential for Infection
Authors: Stoyloff Roman , Strecker Andreas , Bode Liv , Franke Peter , Ludwig Hanns , Hucho Ferdinand
Published: July 2004

Borna disease virus (BDV) is representative of the family of Bornaviridae in the order Mononegavirales (negative‐stranded, non‐segmented, enveloped RNA viruses). It is the causal agent for Borna disease, characterized as an encephalomyelitis (typical form) in a wide variety of domestic animals (from rodents to birds). Recent information shows the involvement of BDV in the pathogenesis of some human psychiatric disorders. The 8.9‐kb viral antigenome codes for five major ORE The third ORF codes for a 16‐kDa protein (matrix protein) that is posttranslationally modified, yielding an N‐linked glycoprotein. Our data show that the glycosylated matrix protein exists as a stable tetrameric structure detectable either by electrospray ionization or matrix‐assisted laser‐desorption ionization mass spectrometry. Under native conditions, the tetramer, with a relative molecular mass of 68 kDa, was isolated from a sediment‐free brain suspension of a BDV‐infected horse. The 68‐kDa entity is stable in the presence of ionic and non‐ionic detergents but dissociates into subunits when heated. We found that the tetrameric matrix protein inhibits in vitro BDV infection in a dose‐dependent manner. In contrast to inhibition of BDV infection with hydrophobic carbohydrate derivatives and protein‐bound glycoconjugates, the glycosylated matrix protein is a very potent inhibitor of BDV infection, indicating that this protein represents an essential virus‐specific membrane component for viral attachment.
Characterization of the Interactions of Alzheimer β‐Amyloid Peptides with Phospholipid Membranes
Authors: McLaurin JoAnne , Chakrabartty Avijit
Published: July 2004

Increasing evidence suggests that Alzheimer β‐amyloid peptides (AAPβ) may be toxic agents in Alzheimer disease. We investigated the possibility that the toxicity may be the result of peptide‐lipid interactions, involving either the cell membrane or the intracellular vesicular system. The interaction of the AAPβ‐(1–40), AAPβ‐(1–42), AAPβ‐(9–25) and AAPβ‐(25–35)‐peptides with acidic and zwitterionic phospholipids was investigated by means of circular dichroism, vesicle disruption and lipid‐aggregation assays. These studies were undertaken at peptide concentrations approaching in vivo levels and at physiological salt concentrations. Circular‐dichroism studies demonstrate that acidic phospholipids induce a conformational change from random coil to β structure in AAPβ‐(1–40)‐peptide and AAPβ‐(1–42)‐peptide at pH 6.0. In contrast, at pH 7.0, only AAPβ‐(1–42)‐peptide was induced to adopt β structure. Phosphatidylinositol was the most efficient inducer of β structure in AAPβ‐(1–42)‐peptide. To further investigate the peptide‐lipid interactions, we examined the ability of the AAPβ peptides to disrupt and/or aggregate phospholipid vesicles. These properties were found to be mediated predominantly through electrostatic interactions with the phospholipid headgroup. The data presented in this paper have implications for AAPβ toxicity and senile‐plaque formation.
Structural Differences in the Crossbridge Head of Temperature‐Associated Myosin Subfragment‐1 Isoforms from Carp Fast Skeletal Muscle
Authors: Hirayama Yasushi , Watabe Shugo
Published: July 2004

We determined the primary structures of the three acclimation‐temperature‐associated isoforms of myosin subfragment‐1 heavy chain from fast skeletal muscle of thermally acclimated carp. These isoforms were cloned by extending 5′‐regions of cDNAs that encode the rod part of myosin heavy chain specifically expressed in 10°C‐ and 30°C‐acclimated carp, together with the region that encodes an intermediate structure [Imai, J., Hirayama, Y., Kikuchi, K., Kakinuma, M. & Watabe, S. (1997) J. Exp. Biol. 200, 27–34]. These three isoforms generally resembled each other in primary structure, showing 94.8, 90.9, and 92% similarity between the 10°C‐ and intermediate‐type, between the 10°C‐ and 30°C‐type, and between the intermediate‐ and 30°C‐type myosin heavy chains, respectively. However, isoform‐specific differences were clearly observed between the 10°C‐ and 30°C‐type heavy chains in the first 60 amino acid residues from the N‐terminus, where the intermediate‐type showed an intermediate feature in its sequence compared to the 10°C‐ and 30°C‐type isoforms. Other striking differences were observed in two surface loops between the 10°C‐ and 30°C‐type isoform. Five amino acid residues out of sixteen were different in loop 1 near the ATP‐binding pocket, and six out of twenty were different in loop 2 on the actin‐binding site. The loops connecting β‐sheets that are known to surround the ATP‐binding pocket were highly conserved in primary structure for the three types. In northern blot analysis, the accumulated mRNA levels of the 10°C‐ and intermediate‐type isoforms were significantly higher in carp acclimated to 10°C and 20 °C than carp acclimated to 30 °C, whereas the level of the 30°C‐type isoform was significantly higher in carp acclimated to 30°C than those acclimated to 10°C and 20°C.
Collagen Fibrillogenesis during Sea Urchin Development
Authors: Lethias Claire , Exposito Jean‐Yves , Garrone Robert
Published: July 2004

The sea urchin 2α fibrillar collagen chain has a unique amino‐propeptide structure with several repetitions of a still unknown 140–145‐amino‐acid, four‐Cys module called SURF (for sea urchin fibrillar module). To follow the expression of the amino‐propeptide of the 2α chain and assign a function to this domain, we have overproduced in Escherichia coli several recombinant proteins corresponding either to the amino‐propeptide or to the amino‐telopeptide. Monoclonal and/or polyclonal antibodies against these recombinant proteins allowed us to observe a similar tissue distribution during the first stages of development. A signal is first observed at the prism stage as intracellular spots in mesenchymal cells. In plutei, immunofluorescence staining is observed around the skeleton spicules and as a thin meshwork surrounding the mesenchymal cells. At the ultrastructural level, and using antibodies against the amino‐propeptide, gold particles are observed at the surface of 25 nm thin periodic fibrils. By rotary shadowing, these fibrils show a brush‐bottle aspect, exhibiting at their surface numerous periodically distributed thin rods ended by a small globule. These data indicate that the amino‐propeptide is maintained during fibrillogenesis. As previously suggested, the retention of the amino‐propeptide could play an important role in regulation of the fibril growth. We propose that the important region of this amino‐propeptide in the widely encountered 25‐nm‐diameter fibrils is the short triple‐helical segment. The globular part of the amino‐propeptide will not only restrict the fibril growth but also interact with other neighbouring components and playing, as suspected from our immunofluorescence studies, a function during the spiculogenesis of the sea urchin embryo.
Identification and Characterization of the Genes for Mitochondrial Ribosomal Proteins of Saccharomyces Cerevisiae
Authors: Kitakawa Madoka , Graack Hanns‐Rüdiger , Grohmann Lutz , Goldschmidt‐Reisin Sabine , Herfurth Elke , Wittmann‐Liebold Brigitte , Nishimura Taisuke , Isono Katsumi
Published: July 2004

We have purified 13 large subunit proteins of the mitochondrial ribosome of the yeast Saccharomyces cerevisiae and determined their partial amino acid sequences. To elucidate the structure and function of these proteins, we searched for their genes by comparing our sequence data with those deduced from the genomic nucleotide sequence data of S. cerevisiae and analyzed them. In addition, we searched for the genes encoding proteins whose N‐terminal amino acid sequences we have reported previously [Grohmann, L., Graack, H.‐R., Kruft, V., Choli, T., Goldschmidt‐Reisin, S. & Kitakawa, M. (1991) FEBS Lett. 284, 51–56]. Thus, we were able to identify and characterize 12 new genes for large subunit proteins of the yeast mitochondrial ribosome. Furthermore, we determined the N‐terminal amino acid sequences of seven small subunit proteins and subsequently identified the genes for five of them, three of which were found to be new.
A Simple Mathematical Model and Practical Approach for Evaluating Citric Acid Cycle Fluxes in Perfused Rat Hearts by 13C‐NMR and 1H‐NMR Spectroscopy
Authors: Tran‐Dinh S. , Hoerter J. A. , Mateo P. , Bouet F. , Herve M.
Published: July 2004

We propose a simple mathematical model and a practical approach for evaluating the flux constant and the absolute value of flux in the citric acid cycle in perfused organs by 13C‐NMR and 1H‐NMR spectroscopy. We demonstrate that 13C‐NMR glutamate spectra are independent of the relative sizes of the mitochondrial and cytosolic compartments and the exchange rates of glutamates, unless there is a difference in 13C chemical shifts of glutamate carbons between the two compartments.

Wistar rat hearts (five beating and four KCl‐arrested hearts) were aerobically perfused with 100% enriched [2‐13C]acetate and the kinetics of glutamate carbon labeling from perchloric acid extracts were studied at various perfusion times. Under our experimental conditions, the citric acid cycle flux constant, which represents the fraction of glutamate in exchange with the citric acid cycle per unit time, is about 0.350 ± 0.003 min−1 for beating hearts and 0.074 ± 0.004 min−1 for KCl‐arrested hearts. The absolute values of the citric acid flux for beating hearts and for KCl‐arrested hearts are 1.06 ± 0.06 μmol · min−1· mg−1 and 0.21 ± 0.02 μmol · min−1· g−1, respectively. The fraction of unlabeled acetate determined from the proton signal of the methyl group is small and essentially the same in beating and arrested hearts (7.4 ± 1.7% and 8.8 ± 2.1%, respectively). Thus, the large difference in the Glu C2/C4 between beating and arrested hearts is not due to the important contribution from anaplerotic sources in arrested hearts but simply to a substantial difference in citric acid cycle fluxes. Our model fits the experimental data well, indicating a fast exchange between 2‐oxoglutarate and glutamate in the mitochondria of rat hearts. Analysis of the flux constant, calculated from the half‐time of glutamate C4 labeling given in the literature, allows for a comparison of the citric acid flux for various working conditions in different animal species.
Biochemical and Cellular Effects of Roscovitine, a Potent and Selective Inhibitor of the Cyclin‐Dependent Kinases cdc2, cdk2 and cdk5
Authors: Meijer Laurent , Borgne Annie , Mulner Odile , Chong James P. J. , Blow J. Julian , Inagaki Naoyuki , Inagaki Masaki , Delcros Jean‐Guy , Moulinoux Jacques‐Philippe
Published: July 2004

Cyclin‐dependent kinases (cdk) play an essential role in the intracellular control of the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. Enzymatic screening has recently led to the discovery of specific inhibitors of cyclin‐dependent kinases, such as butyrolactone I, flavopiridol and the purine olomoucine. Among a series of C2, N6, N9‐substituted ade‐nines tested on purified cdc2/cylin B, 2‐(1‐ethyl‐2‐hydroxyethylamino)‐6‐benzylamino‐9‐isopropylpu‐rine (roscovitine) displays high efficiency and high selectivity towards some cyclin‐dependent kinases. The kinase specificity of roscovitine was investigated with 25 highly purified kinases (including protein kinase A, G and C isoforms, myosin light‐chain kinase, casein kinase 2, insulin receptor tyrosine kinase, c‐src, v‐abl). Most kinases are not significantly inhibited by roscovitine. cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdkSlp35 only are substantially inhibited (IC50values of 0.65, 0.7, 0.7 and 0.2 μM, respectively). cdk4/cyclin D1 and cdk6/cyclin D2 are very poorly inhibited by roscovitine (IC50>100 μM). Extracellular regulated kinases erkl and erk2 are inhibited with an IC50of 34 μM and 14 μM, respectively. Roscovitine reversibly arrests starfish oocytes and sea urchin embryos in late pro‐phase. Roscovitine inhibitsin vitroM‐phase‐promoting factor activity andin vitroDNA synthesis inXenopusegg extracts. It blocks progesterone‐induced oocyte maturation ofXenopusoocytes andin vivophosphorylation of the elongation factor eEF‐1. Roscovitine inhibits the proliferation of mammalian cell lines with an average IC50] of 16 μM. In the presence of roscovitine L1210 cells arrest in G1 and accumulate in G2.In vivophosphorylation of vimentin on Ser55 by cdc2/cyclin B is inhibited by roscovitine. Through its unique selectivity for some cyclin‐dependent kinases, roscovitine provides a useful anti‐mitotic reagent for cell cycle studies and may prove interesting to control cells with deregulated cdc2, cdk2 or cdk5 kinase activities.
Structure and Function of a Second Gene Cluster Encoding the Formate Dehydrogenase of Wolinella Succinogenes
Authors: Lenger Ruth , Herrmann Ute , Gross Roland , Simon Jörg , Kröger Achim
Published: July 2004

Wolinella succinogenes contains a single formate dehydrogenase, but two gene loci (fdhI and fdhII) code for the subunits of the enzyme. The nucleotide sequence of fdhII is almost identical with that of fdhI in the region comprising fdhEABCD. The sequences of fdhI and fdhII differ in the promoter regions upstream of fdhE. Deletion mutants lacking either fdhI or fdhII synthesize functional formate dehydrogenases, as shown by growth with formate as electron donor and either fumarate or polysulfide as acceptor substrates, and by the presence of the FdhA subunit and of enzyme activity. In the wild‐type strain, the fdhI genes appear to be expressed preferentially during growth with formate and fumarate. The six‐times greater amount of the enzyme present upon growth with formate and polysulfide is due to the expression of both fdhI and fdhII. The transcription start sites were located 196‐bp and 129‐bp upstream of the fdhE start codons of fdhI and fdhII, respectively. An apparently single transcript (5.6 kbp) was detected in polysulfide‐grown W. succinogenes by Northern‐blot analysis, suggesting that the five open reading frames form operons.
Pathway of Detergent‐Mediated and Peptide Ligand‐Mediated Refolding of Heterodimeric Class II Major Histocompatibility Complex (MHC) Molecules
Authors: Stöckel Johannes , Döring Klaus , Malotka Joachim , Jähnig Fritz , Dornmair Klaus
Published: July 2004

We investigated the mechanism of refolding and reassembly of recombinant α and β chains of the class II major histocompatibility molecules (MHC‐II) HLA‐DRB5*0101. Both chains were expressed in the cytosol of Escherichin coli, purified in urea and SDS, and reassembled to functional heterodimers by replacement of SDS by mild detergents, incubation in a redox‐shuffling buffer and finally by oxidation and removal of detergent. Refolding was mediated by mild detergents and by peptide ligands. Early stages of structure formation were characterized by circular dichroism, fluorescence, and time‐resolved fluorescence anisotropy decay (FAD) spectroscopies. We found that formation of secondary structure was detectable after replacement of SDS by mild detergents. At that stage the α and β chains were still monomeric, the buffer was strongly reducing, and the folding intermediates did not yet interact with peptide ligands. Formation of folding intermediates capable of interacting with peptide ligands was detected after adjusting the redox potential with oxidized glutathione and incubation in mild detergents. We conclude that at that stage a tertiary structure close to the native structure is formed at least locally. The nature and concentration of detergent critically determined the refolding efficiency. We compared detergents with different carbohydrate headgroups, and with aliphatic chains ranging from C6 to C14 in length. For each of the detergents we observed a narrow concentration range for mediating refolding. Surprisingly, detergents with long aliphatic chains had to be used at higher concentrations than short‐chain detergents, indicating that increasing the solubility of folding intermediates is not the only function of detergents during a refolding reaction. We discuss structure formation and interactions of detergents with stable folding intermediates. Understanding such interactions will help to develop rational strategies for refolding hydrophobic or oligomeric proteins.
The Effects of High Salt Concentrations on the Regulation of the Substrate Specificity of Human Recombinant Deoxycytidine Kinase
Authors: Usova Elena V. , Eriksson Staffan
Published: July 2004

Deoxycytidine kinase (dCK) is a salvage pathway enzyme with broad substrate specificity that can phosphorylate both pyrimidine and purine deoxynucleosides, including important antiviral and cytostatic agents. The kinetic behaviour of dCK is complex with saturation curves showing negative cooperativity. In this study, we have expressed and affinity purified recombinant dCK, using the PET 9d vector system with a histidine tag‐sequence and a thrombin cleavage site fused to the N‐terminus of the dCK coding sequence. The His‐tagged protein showed essentially the same kinetic properties as the protease cleaved protein and the purified protein isolated from human spleen. However, addition of 0.2–0.4 M NaCl during the dCK reaction caused a stimulation of 2′‐deoxycytidine (dCyd), and the antileukemic analog 2‐chlorodeoxyadenosine (CldAdo) phosphorylation, but an inhibition of the 2′‐deoxyguanosine (dGuo) phosphorylation, both with His‐tagged and protease cleaved dCK. The negative cooperativity observed with dCyd was eliminated by the presence of 0.4 M NaCl so that the Hill coefficient changed from 0.6 to 1.4. In contrast, dGuo phosphorylation that initially followed Michaelis‐Menten kinetics showed negative cooperativity after addition of 0.4 NaCl. The alterations in kinetic properties were not accompanied by any apparent changes in subunit structure as revealed by gel filtration. The major form of dCK eluted in the position corresponding to a dimer in the presence or absence of salt, but a minor fraction of dCK, eluting in the position of a tetramer, was diminished in the presence of salt. The mechanism for the effects of 0.4 M NaCl on dCK kinetic behaviour is not known but it is most likely due to alterations in the conformation of the active site of the enzyme. The effects described here also may explain some of the discrepancies reported in the literature on the substrate specificity of this complex enzyme.
The Stathmin Family
Authors: Ozon Sylvie , Maucuer Alexandre , Sobel André
Published: July 2004

Stathmin is a ubiquitous phosphoprotein proposed to be a relay integrating various intracellular signaling pathways. Its high phylogenetic conservation and the identification of the related molecules, SCG10 in rat and XB3 in Xenopus, suggested the existence of a stathmin‐related family. A systematic PCR‐based approach allowed the identification of several novel mammalian sequences of which two coded for expressed members of the stathmin family; the translated RB3 sequence shares 88% amino‐acid identity with that of XB3 and is thus its rat homologue, and RB3′ corresponds to an alternatively spliced product of the same gene, encoding a truncated form. Within their stathmin‐like domain, the α helix, probably responsible for coiled‐coil protein‐protein interactions, is conserved, as well as are two consensus phosphorylation sites; in their N‐terminal extension domain, two cystein residues most likely responsible for membrane attachment through palmitoylation, are present in RB3/RB3′ as in SCG10. The novel identification and characterization of the corresponding proteins showed that all three are associated with the particulate, membrane‐containing fraction. They furthermore display several spots of decreasing pI on two‐dimensional immunoblots, suggesting that they are phosphorylated in vivo. As for SCG10, RB3 mRNA is detectable only in the nervous system by in situ hybridization, but at similar levels in the newborn and the adult brain as revealed by Northern blots, whereas SCG10 expression decreases in the adult. Furthermore, RB3 mRNA is undetectable in PC12 cells, whereas SCG10 mRNA increases after treatment with nerve growth factor, inducing neuronal differentiation. In conclusion, we demonstrate here the existence of a highly conserved stathmin‐related family in mammals, of which each member seems to play specific roles, related to the control of cell proliferation and activities for stathmin and to that of neuronal differentiation for SCG10, the novel RB3/RB3′ proteins being rather related to the expression of differentiated neuronal functions.
The Catalytic Subunit of Dictyostelium cAMP‐Dependent Protein Kinase
Authors: Etchebehere Lilian C. , Van Bemmelen Miguel X. P. , Anjard Christophe , Traincard François , Assemat Karine , Reymond Christophe , Veron Michel
Published: July 2004

The C subunit of Dictyostelium cAMP‐dependent protein kinase (PKA) is unusually large (73 kDa) due to the presence of 330 amino acids N‐terminal to the conserved catalytic core. The sequence following the core, including a C‐terminal‐Phe‐Xaa‐Xaa‐Phe‐COOH motif, is highly conserved. We have characterized the catalytic activity and stability of C subunits mutated in sequences outside the catalytic core and we have analyzed their ability to interact with the R subunit and with the heat‐stable protein‐kinase inhibitor PKI.

Mutants carrying deletions in the N‐terminal domain displayed little difference in their kinetic properties and retained their capacity to be inhibited by R subunit and by PKI. In contrast, the mutation of one or both of the phenylalanine residues in the C‐terminal motif resulted in a decrease of catalytic activity and stability of the proteins. Inhibition by the R subunit or by PKI were however unaffected. Sequence‐comparison analysis of other protein kinases revealed that a‐Phe‐Xaa‐Xaa‐Phe‐motif is present in many Ser/Thr protein kinases, although its location at the very end of the polypeptide is a particular feature of the PKA family. We propose that the presence of this motif may serve to identify isoforms of protein kinases.
The Structure, Function and Application of the Hammerhead Ribozyme
Authors: Birikh Klara R. , Heaton Paul A. , Eckstein Fritz
Published: July 2004

The hammerhead ribozyme is one of the smallest ribozymes known and catalyses the site‐specific hydrolysis of a phosphodiester bond. This small ribozyme is of interest for two reasons. It offers a convenient system to study the structure/function relationship of a nucleotide sequence, and is a potential vehicle for the inhibition of gene expression. The first part of the review summarizes the sequence requirements of the hammerhead, its three‐dimensional structure and the proposed mechanism, in addition to ribozyme specificity and turnover. The second part of the review focuses on the in vivo application of the ribozyme. The processes involved in designing ribozymes for efficient cleavage in vivo are described, together with possible delivery strategies.
Linking Protein Kinase C to Cell‐Cycle Control
Authors: Livneh Etta , Fishman Daniel D.
Published: July 2004

Protein kinase C (PKC) isoenzymes are involved in diverse cellular functions, including differentiation, growth control, tumor promotion, and cell death. In recent years, evidence has began to emerge suggesting a role for PKC in cell cycle control. A paper published recently, demonstrating a functional link between PKC and cell cycle control in yeast (Marini, N. J., Meldrum, E., Buehrer, B., Hubberstey, A. V., Stone, D. E., Traynor‐Kaplan, A. & Reed, S. I. (1996) EMBO J. 15, 3040–3052), strengthens this data. Thus, the existence of cell‐cycle‐regulated pathways involving PKC in both yeast and mammals indicate that PKC may be a conserved regulator of cell cycle events that links signal transduction pathways and the cell‐cycle machinery. In this paper, we will review current data on the cell cycle components that are targets for PKC regulation.

PKC enzymes appear to operate as regulators of the cell cycle at two sites, during G1 progression and G2/M transition. In G1, the overall effect of PKC activation is inhibition of the cell cycle at mid to late G1. This cell cycle inhibition correlates with a blockage in the normal phosphorylation of the tumor suppressor retinoblastoma Rb protein, presumably through an indirect mechanism. The reduced activity of the cyclin‐dependent kinase, Cdk2, appears to be the major effect of PKC activation in various cell systems. This may also underlie the inhibition of Rb phosphorylation exhibited by PKC activation. Several mechanisms were described in different studies on the regulation of Cdk2 activity by PKC; reduced Cdk‐activating kinase activity, diminished expression of the Cdk2 partners cyclins E or A, and the increased expression of the cyclin‐dependent inhibitors, p21WAF1 and p27KIPI, which are capable of binding to cyclin/Cdk2 complexes.

PKC enzymes were also shown to play a role in G2/M transition. Among the suggested mechanisms is suppression of Cdc2 activity. However, most of the published data strongly implicate PKC in lamin B phosphorylation and nuclear envelope disassembly.
Recognition and Cleavage of DNA by Type‐II Restriction Endonucleases
Authors: Pingoud Alfred , Jeltsch Albert
Published: July 2004

Restriction endonucleases are enzymes which recognize short DNA sequences and cleave the DNA in both strands. Depending on the enzymological properties different types are distinguished. Type II restriction endonucleases are homodimers which recognize short palindromic sequences 4–8 bp in length and, in the presence of Mg2+, cleave the DNA within or next to the recognition site. They are capable of non‐specific binding to DNA and make use of linear diffusion to locate their target site. Binding and recognition of the specific site involves contacts to the bases of the recognition sequence and the phosphodiester backbone over approximately 10–12 bp. In general, recognition is highly redundant which explains the extreme specificity of these enzymes. Specific binding is accompanied by conformational changes over both the protein and the DNA. This mutual induced fit leads to the activation of the catalytic centers. The precise mechanism of cleavage has not yet been established for any restriction endonuclease. Currently two models are discussed: the substrate‐assisted catalysis mechanism and the two‐metal‐ion mechanism. Structural similarities identified between EcoRI, EcoRV, BamHI, PvuII and Cfr10I suggest that many type II restriciton endonucleases are not only functionally but also evolutionarily related.
Distribution of the lipolysis stimulated receptor in adult and embryonic murine tissues and lethality of LSR–/– embryos at 12.5 to 14.5 days of gestation
Authors: Mesli Samir , Javorschi Sandrine , Bérard Annie M. , Landry Marc , Priddle Helen , Kivlichan David , Smith Andrew J. H. , Yen Frances T. , Bihain Bernard E. , Darmon Michel
Published: July 2004

The lipolysis stimulated receptor (LSR) recognizes apolipoprotein B/E‐containing lipoproteins in the presence of free fatty acids, and is thought to be involved in the clearance of triglyceride‐rich lipoproteins (TRL). The distribution of LSR in mice was studied by Northern blots, quantitative PCR and immunofluorescence. In the adult, LSR mRNA was detectable in all tissues tested except muscle and heart, and was abundant in liver, lung, intestine, kidney, ovaries and testes. During embryogenesis, LSR mRNA was detectable at 7.5 days post‐coitum (E7) and increased up to E17 in parallel to prothrombin, a liver marker. In adult liver, immunofluorescence experiments showed a staining at the periphery of hepatocytes as well as in fetal liver at E12 and E15. These results are in agreement with the assumption that LSR is a plasma membrane receptor involved in the clearance of lipoproteins by liver, and suggest a possible role in steroidogenic organs, lung, intestine and kidney). To explore the role of LSR in vivo, the LSR gene was inactivated in 129/Ola ES cells by removing a gene segment containing exons 2–5, and 129/Ola‐C57BL/6 mice bearing the deletion were produced. Although heterozygotes appeared normal, LSR homozygotes were not viable, with the exception of three males, while the total progeny of genotyped wild‐type and heterozygote pups was 345. Mortality of the homozygote embryos was observed between days 12.5 and 15.5 of gestation, a time at which their liver was much smaller than that of their littermates, indicating that the expression of LSR is critical for liver and embryonic development.
Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungusTalaromyces emersonii
Authors: Maloney Alan P. , Callan Susan M. , Murray Patrick G. , Tuohy Maria G.
Published: July 2004

Mitochondrial malate dehydrogenase (m‐MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungusTalaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m‐MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52 °C in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m‐MDHs than homologs from invertebrate or mesophilic fungal sources. The full‐length m‐MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N‐terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m‐MDH genes. The m‐MDH gene is the first oxidoreductase gene cloned fromT. emersoniiand is the first full‐length m‐MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote. Recombinant m‐MDH was expressed inEscherichia coli, as a His‐tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni2+‐nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture. The recombinant enzyme behaved as a dimer under nondenaturing conditions. Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His‐tag. Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme.
Characterization of a Cry1Ac‐receptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae
Authors: Jurat‐Fuentes Juan L. , Adang Michael J.
Published: July 2004

We reported previously a direct correlation between reduced soybean agglutinin binding to 63‐ and 68‐kDa midgut glycoproteins and resistance to Cry1Ac toxin from Bacillus thuringiensis in the tobacco budworm (Heliothis virescens). In the present work we describe the identification of the 68‐kDa glycoprotein as a membrane‐bound form of alkaline phosphatase we term HvALP. Lectin blot analysis of HvALP revealed the existence of N‐linked oligosaccharides containing terminal N‐acetylgalactosamine required for [125I]Cry1Ac binding in ligand blots. Based on immunoblotting and alkaline phosphatase activity detection, reduced soybean agglutinin binding to HvALP from Cry1Ac resistant larvae of the H. virescens YHD2 strain was attributable to reduced amounts of HvALP in resistant larvae. Quantification of specific alkaline phosphatase activity in brush border membrane proteins from susceptible (YDK and F1 generation from backcrosses) and YHD2 H. virescens larvae confirmed the observation of reduced HvALP levels. We propose HvALP as a Cry1Ac binding protein that is present at reduced levels in brush border membrane vesicles from YHD2 larvae.
Structural and functional analysis of ataxin‐2 and ataxin‐3
Authors: Albrecht Mario , Golatta Michael , Wüllner Ullrich , Lengauer Thomas
Published: July 2004

Spinocerebellar ataxia types 2 (SCA2) and 3 (SCA3) are autosomal‐dominantly inherited, neurodegenerative diseases caused by CAG repeat expansions in the coding regions of the genes encoding ataxin‐2 and ataxin‐3, respectively. To provide a rationale for further functional experiments, we explored the protein architectures of ataxin‐2 and ataxin‐3. Using structure‐based multiple sequence alignments of homologous proteins, we investigated domains, sequence motifs, and interaction partners. Our analyses focused on presumably functional amino acids and the construction of tertiary structure models of the RNA‐binding Lsm domain of ataxin‐2 and the deubiquitinating Josephin domain of ataxin‐3. We also speculate about distant evolutionary relationships of ubiquitin‐binding UIM, GAT, UBA and CUE domains and helical ANTH and UBX domain extensions.