bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Determination of the Regulation of Oxidative Phosphorylation by Cadmium in Potato Tuber Mitochondria
Authors: Kesseler Adolf , Brand Martin D.
Published: March 2005

The effects of cadmium on respiration rate, phosphorylation rate, proton leak rate, the protonmotive force and the effective P/O ratio were determined over a range of respiratory conditions and cadmium concentrations by applying top‐down regulation analysis. To quantify the effects of cadmium, we determined the overall response coefficients of these variables of oxidative phosphorylation to cadmium in different respiratory states between state 4 and state 3 and at different cadmium concentrations. The overall response coefficients to cadmium showed quantitatively how cadmium stimulated substrate oxidation rate at high cadmium concentrations near state 4 but inhibited it to different extents under all other conditions, how cadmium inhibited the rate of proton leak rate at low cadmium concentrations near state 4 but stimulated it under all other conditions, and how cadmium inhibited the rate of phosphorylation and depressed the protonmotive force and the effective P/O ratio to different extents under all conditions. Cadmium is known to stimulate the proton leak and to inhibit the substrate oxidation reactions; we calculated the elasticities of these subsystems to cadmium to quantify its effects. To describe fully how the cadmium effects on different subsystems produce the overall responses of the system to cadmium, we then calculated the partial response coefficients of the system variables to cadmium acting through each subsystem. The partial response coefficients quantify the contribution of each block to the overall effect of cadmium on each variable in any condition, and sum to the overall response coefficient in each condition. Together with the elasticity analysis and the control analysis and internal regulation analysis presented in the preceding papers [Kesseler, A. & Brand, M. D. (1994) Eur. J. Biochem. 225, pp. 897–906; Kesseler, A. & Brand, M. D. (1994) Eur. J. Biochem. 225, pp. 907–9221 they completely describe how cadmium exerts its effects on oxidative phosphorylation at the system level.
Characterization of a Rat C6 Glioma‐Secreted Follistatin‐Related Protein (FRP)
Authors: Zwijsen An , Blockx Herman , Van Arnhem Wouter , Willems Jean , Fransen Lucie , Devos Kathleen , Raymackers Jos , Van De Voorde André , Slegers Herman
Published: March 2005

A protein was isolated from rat C6 glioma‐conditioned medium and was biochemically characterized. The heparin‐binding protein has a native molecular mass of 55–75000Da, a molecular mass of 40–48000 Da under denaturing conditions, and a pI of 5.0–6.0. Based on the determined partial amino acid sequences, the full lenght cDNA encoding the rat and human proteins were cloned. The cDNA sequences identified the isolated rat and human protein as the homologue of a recently reported mouse osteoblast‐transforming‐growth‐factor‐β1‐inducible protein, encoded by the TSC‐36 gene [Shibanuma, M., Mashimo, J., Mita, A., Kuroki, T. & Nose, K. (1993) Eur. J. Biochem. 217, 13–19].

Analysis of the human, rat and mouse amino acid sequences indicates that these proteins are highly conserved (>92% sequence identity). Sequence similarities with follistatin and the follistatin‐like domain of agrin are revealed. The relationship with follistatin and agrin points to possible common functions for the cloned follistatin‐related proteins (FRP).

The protein has no effect on the inhibitory action of transforming growth factor‐β1, on CCl‐64 cell growth.
Dexamethasone and Insulin Demonstrate Marked and Opposite Regulation of the Steady‐State mRNA Level of the Peroxisomal Proliferator‐Activated Receptor (PPAR) in Hepatic Cells
Authors: Steineger Hilde Hermansen , Sørensen Hilde Nebb , Tugwood Jonathan D. , Skrede Steinar , Spydevold Øystein , Gautvik Kaare M.
Published: March 2005

Fatty acids and the peroxisomal proliferator, 3‐tetradecylthioacetic acid (TTA) stimulate transcription of peroxisomal β‐oxidation enzymes. Recently, we have shown that their actions are markedly modulated by dexamethasone and insulin which show synergistic and inhibitory effects, respectively. In this study, we describe the regulation of the peroxisomal proliferator‐activated receptor (PPAR), a member of the steroid‐hormone‐receptor superfamily, in a similar manner by hormones and fatty acids, supporting the hypothesis that PPAR may act as a ligand‐activated transcription factor. Northern‐blot analysis of steady‐state mRNA levels revealed three different specific transcripts for PPAR of 10.2, 4.6 and 1.8 kb, and the former two being regulated in hepatic tissue, hepatocytes and hepatoma cells. Dexamethasone produced a pronounced overall stimulatory effect (15.3‐fold) in rat hepatocytes, while insulin blocked this action completely. Minor inductions of PPAR mRNA (up to twofold induction) were observed when different fatty acids were administrated alone. However, in combination with dexamethasone, additive or synergistic actions, mounting to 24‐fold stimulation, were observed, while insulin always exerted an over‐riding down‐regulatory effect. In non‐fasting rats receiving dexamethasone, elevation of serum insulin, a slight increase in serum free fatty acids accompanied by PPAR mRNA level increases of 2.4‐fold and stimulation of liver peroxisomal acyl‐CoA oxidase mRNA were observed. Our results suggest that PPAR mRNA expression is under strict hormonal control and that the fatty acids and hormones affect PPAR mRNA levels in a manner analogous to the regulation of the peroxisomal β‐oxidation enzymes. The PPAR gene‐regulating unit apparently contains hormone‐response elements (HRE) for dexamethasone and insulin, which are thus functionally important for PPAR transcription in liver cells, making a significant enhancement or inhibition of the physiological actions of fatty acids possible.
Binding of Purified Collagen Receptors (α1β1, α2β1) and RGD‐Dependent Integrins to Laminins and Laminin Fragments
Authors: Pfaff Martin , Göhring Walter , Brown Judith C. , Timpl Rupert
Published: March 2005

Integrins α1β1 and α2β1, when purified by collagen affinity chromatography, showed distinct binding to mouse tumor laminin‐1, which has the chain composition α1β1γ1. The binding was, however, about 10‐fold lower than to collagen IV. Only little (α1β1) or no binding (α2β1) was observed to two different laminin isoforms (α2β1γ1, α2β2γ1) from human placenta. Binding to laminin‐1 was abolished by EDTA and could be specifically inhibited by antibodies to the respective integrin a subunit. These antibodies also inhibited cell adhesion to collagens. The binding of soluble integrins was weaker than that of immobilized integrins but could be enhanced by an activating anti(β1 integrin). No enhancement was observed for immobilized integrins. Studies with laminin‐1 fragments demonstrated lack of binding to the major cell‐adhesive fragment E8 from the long arm, fragments E3 and E4, involved in heparin‐binding and self‐assembly, respectively, and fragment P1, corresponding to the inner segments of the short arms. A larger short‐arm fragment (E1XNd), which lacks the N‐terminal β1 chain domains V and VI, was as active as laminin. Together, these results, suggested the localization of the binding sites for α1β1 and α2β1 to the N‐terminal region of the laminin α1 chain. Fragment P1 but not intact laminin‐1 bound to αVβ3 integrin in an EDTA‐sensitive and RGD‐sensitive manner, underscoring previous data on the cryptic nature of the RGD site in laminin‐1. Further analyses by surface plasmon resonance assays demonstrated a KD= 50 nM for α2β1/laminin‐1 binding and a KD= 450 nM for αVβ3/fragment P1 binding and confirmed the anti‐β1‐mediated increase in affinity for α2β1.
Isolation and Structural Analysis of Oligosaccharide Phosphates Containing the Complete Carbohydrate Chain of the Lipopolysaccharide from Vibrio cholerae Strain H11 (Non‐O1)
Authors: Bock Klaus , Vinogradov Evgeny V. , Holst Otto , Brade Helmut
Published: March 2005

For the first time, an oligosaccharide has been prepared comprising the lipid A backbone, the core oligosaccharide and one repeating unit of the O‐specific polysaccharide (O‐chain) of a lipopolysaccharide. Lipopolysaccharide from Vibrio cholerae strain H11 (non‐O1) was deacylated and the products were separated by high‐performance anion‐exchange chromatography. Major fractions were a hexadecasaccharide trisphosphate 1, representing the core‐lipid A oligosacccharide substituted by one modified repeating unit of the O‐antigenic polysaccharide, a dodecasaccharide trisphosphate 2 and an undecasaccharide trisphosphate 3, representing the core‐lipid A region. Oligosaccharide 1 originated from β‐elimination upon alkaline hydrolysis of α‐galacturonic acid of the O‐chain; oligosaccharides 2 and 3 were most likely obtained from naturally occurring lipopolysaccharide species carrying no O‐chain. The structures of these compounds were elucidated on the basis of monosaccharide composition, and NMR investigations comprising correlation spectroscopy, total correlation spectroscopy and nuclear Overhauser enhancement spectroscopy experiments, as well as heteronuclear 13C,1H correlation spectroscopy. The structures are as follows:

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where R is β L‐threo ‐hex‐4‐enuronopyranosyl‐(1–4)‐α‐Neu‐(2–3)‐β‐GalA‐(1–3)‐β‐QuiN‐(1–4)‐β‐Sedf ‐(2‐ in 1, β‐Sedf ‐(2‐ in 2, and H in 3. Where not stated otherwise, sugars are pyranoses of the D‐series. Hep is L‐glycero ‐D‐manno ‐heptose, QuiN is 2‐amino‐2,6‐dideoxy‐glucose, Kdo is 3‐deoxy‐D‐manno ‐2‐octulosonic acid, Sed is D‐altro ‐heptulose and GalA is galacturonic acid.
Effect of Basic Fibroblast Growth Factor on Synthesis/Secretion of pS2 Protein by Human Breast Cancer Cells (MCF‐7)
Authors: Miyashita Sadakazu , Hirota Masao , Yamamoto Toshiya , Shiroyama Chiyoka , Furukawa Yoshiko , Hayashi Kyozo
Published: March 2005

pS2 is an estrogen‐induced mRNA species that was originally identified in the breast cancer cell line MCF‐7. Exposure of the cells to basic fibroblast growth factor (bFGF) at the concentration of 10–100 ng/ml for 48–72 h resulted in a marked increase in the concentration of pS2 protein in the medium. The polymerase chain reaction with reverse transcriptase revealed that bFGF increased the amount of intracellular pS2 mRNA; immunocytochemical studies showed that exposure to the factor increased the amount of intracellular pS2 protein. Simultaneous addition of cycloheximide with bFGF completely abolished induction of pS2 protein, although it did not affect the induction of pS2 mRNA. Actinomycin D did not affect the stimulatory effect of bFGF on synthesis/secretion of pS2 protein. bFGF effectively abolished decay of the pS2 mRNA level caused by actinomycin D. These results suggest that the induction of the synthesis/secretion of pS2 protein by bFGF occurs at the post‐transcriptional level, most probably due to the stabilization of pS2 mRNA. Another finding, that bFGF and estradiol have a synergistic effect on induction of pS2 protein, suggests the possibility that these two inducers act by a different but partly overlapping mechanism.
Epidermal‐Growth‐Factor‐Dependent Activation of the Src‐Family Kinases
Authors: Osherov Nir , Levitzki Alexander
Published: March 2005

The precise role of src‐type kinases as signal transducers has been under intensive investigation but only in a few instances has their role been revealed in any detail. Thus, src, fyn and yes are activated upon stimulation by platelet‐derived growth factor or colony‐stimulating factor in cells expressing high levels of these receptors. Activation of src‐family kinases by other receptor tyrosine kinases such as the epidermal‐growth‐factor (EGF) receptor has not been directly demonstrated. In this report, we demonstrate EGF‐dependent activation of src‐family tyrosine kinases in NIH3T3 cells overexpresssing the human EGF receptor. Activation is rapid (<1 min) and persistent (up to 16 h). Furthermore, we show a correlation between the level of EGF receptor expressed and the degree of src‐family kinase activation. We show that src‐family kinase activity is also activated by addition of EGF to PC12 cells, which endogenously express relatively high levels of EGF receptor. Most strikingly, we show that A431 cells, which endogenously express very high levels of EGF receptor, show 10‐fold elevated src‐family kinase activity as compared to DHER14 cells, and that this activity is constitutive. This activity is completely blocked by AG1478, a specific inhibitor of the EGF‐receptor tyrosine kinase activity, pointing to a direct link between overexpression of the EGF receptor and enhanced src‐family kinase activity. Our findings suggest that EGF‐dependent src‐family kinase activity is detectable only when the levels of EGF receptor reach a specific level. Additionally, high levels of EGF receptor, as in A431 cells, may contribute to the elevated activation of src‐family kinases. Sustained src‐family kinase activation, similar to that seen in v‐src‐trans‐formed cells, may play a role in tumorogenesis and tumor maintenance.
Familial Alzheimer's Disease Cells Abnormally Accumulate β‐Amyloid‐Harbouring Peptides Preferentially in Cytosol but not in Extracellular Fluid
Authors: Matsumoto Akira , Matsumoto Reiko
Published: March 2005

The proteolytic processing of β‐amyloid precursor protein (APP) was analyzed and compared for familial Alzheimer's disease and normal lymphoid cells, focusing on β‐amyloid‐harbouring peptides and the extracellular fragments released into the medium. Tris/tricine gel electrophoresis of anti‐βA4‐(8–17)‐immunoprecipitated peptides and subsequent N‐terminal amino acid sequencing revealed previously unidentified peptides; the 14‐kDa peptide with a βA4 N‐terminus and the 12‐kDa peptide with an Esch's‐site N‐terminus in the cytosols, and the same 12‐kDa peptide predominating in the media. Moreover, some early onset familial Alzheimer's disease cells, but not normal cells, express a 4‐kDa peptide with a β‐amyloid N‐terminus in the cytosol. Two‐dimensional gel electrophoresis of the extracellular APP peptides immunoprecipitated with anti‐APP‐(144–654)‐peptide showed that familial Alzheimer's disease cells are deficient in processing, especially the 50–53‐kDa peptides with the Kunitz‐protease‐inhibitor domain. This may reflect their unique expression of a serine protease identified as cleaving APP at the β‐amyloid N‐terminus.
Metabolic Relationship Between the CO Dehydrogenase Molybdenum Cofactor and the Excretion of Urothione by Hydrogenophaga pseudoflava
Authors: Volk Matthias , Meyer Ortwin , Frunzke Kurt
Published: March 2005

Urothione was isolated as an excretion product of Hydrogenophaga pseudoflava and other bacteria at amounts approaching 253 μg/l of culture corresponding to 44 μg/g bacterial dry mass. The compound was identified as urothione by co‐chromatography with urothione isolated from human urine, its characteristic ultraviolet and visible absorption spectra, oxidation to pterin‐6‐carboxylic‐7‐sulfonic acid by alkaline permanganate, 1H‐NMR spectroscopy, double‐quantum‐filtered Fourier‐transform 1H correlated spectroscopy, circular‐dichroism spectroscopy and mass spectroscopy. A metabolic relationship between urothione and the carbon monoxide dehydrogenase molybdenum cofactor was suggested by a 1.1:0.5 molar ratio between urothione excreted and degradation of carbon monoxide dehydrogenase, a coincidence of urothione excretion and induction of carbon monoxide dehydrogenase with different species of carboxidotrophic bacteria, a structural relationship between molybdopterin cytosine dinucleotide of the carbon monoxide dehydrogenase molybdenum cofactor and urothione, and the demonstrated conversion of the carbon monoxide dehydrogenase molybdenum cofactor to urothione in vitro. A pathway for the conversion of the H. pseudoflava carbon monoxide dehydrogenase molybdenum cofactor to urothione has been proposed which involves molybdopterin cytosine dinucleotide, molybdopterin, phospho‐norurothione and norurothione.
Tandemly Arranged Repeats of a Novel Highly Charged 16–Amino‐Acid Motif Representing the Major Component of the Sperm‐Tail‐Specific Axoneme‐Associated Protein Family Dhmst101 form Extended α‐Helical Rods within the Extremely Elongated Spermatozoa of Drosophila hydei
Authors: Neesen Jürgen , Padmanabhan S. , Bünemann Hans
Published: March 2005

We have previously reported that the sperm‐tail‐specific proteins in Drosophila hydei, encoded by the small Dhmst101 gene family, contained several tandem repeats of a novel highly charged, well‐conserved cysteine‐containing motif of 16 amino acids, KKKCAEAAKKEKEAAE [Neesen, J., Bünemann, H. & Heinlein, U. A. O. (1994) Dev. Biol. 162, 414–425] and suggested that this motif might be important in the structural and functional integrity of the sperm tail. We tested this suggestion by examining structure formation by model synthetic peptides containing the 16‐residue sequence and corresponding peptides with one and two repeats of the sequence with Cys being replaced by Ala. We find that all these peptides form monomeric α‐helices and that the helix content is considerably enhanced as the number of tandem repeats increases. These results are consistent with tandemly arranged 16‐amino‐acid repeats in Dhmst101 proteins forming extended α‐helical rods, with the highly conserved Cys present in each 16‐amino‐acid motif being involved in regular interhelical cross‐linking, thus providing a rigid, stable framework within the extremely elongated spermatozoa of Drosophila hydei.
Kinetics of the Two Forms of Acetyl‐CoA Carboxylase from Pisum sativum
Authors: Dehaye Laure , Alban Claude , Job Claudette , Douce Roland , Job Dominique
Published: March 2005

Steady‐state kinetics of the 220‐kDa form of acetyl‐CoA carboxylase (ACC220), as purified from mature pea seeds, have been investigated with respect to the substrate specificity and inhibition by quizalofop, a herbicide of the aryloxyphenoxypropionate type. The enzyme showed a dual specificity, being able to carboxylate propionyl‐CoA at a maximal rate approximately 20% that measured in the presence of the acetyl‐CoA substrate. These two reactions occur at separate sites on the enzyme. One site binds either acetyl‐CoA (Km= 226 μM) or propionyl‐CoA (Km= 38 μM) and is strongly inhibited by quizalofop (Ki= 25 μM and 9.3 μM for the acetyl‐CoA and propionyl‐CoA substrates, respectively). The other is specific for acetyl‐CoA (Km= 11 μM) and is much less inhibited by quizalofop (Ki= 256 μM). Owing to the existence of these two catalytically different sites, the enzyme obeyed Michaelis‐Menten kinetics with propionyl‐CoA, but exhibited kinetic cooperativity in the presence of acetyl‐CoA. Also, kinetics of propionyl‐CoA carboxylase activity of ACC220 exhibited hyperbolic inhibition in the presence of quizalofop, but co‐operative inhibition when following the ACC activity of the enzyme. The results suggest that the higher the substrate specificity, the lower the quizalofop sensitivity of the active site. Similar kinetic behaviour was observed with ACC220 purified from pea leaves. Also, the apparent correlation between the substrate specificity and the sensitivity of ACC towards quizalofop was confirmed by kinetic analyses of the low‐molecular‐mass form of ACC present in chloroplasts of young pea leaves. This enzyme was insensitive to quizalofop inhibition and was not able to carboxylate propionyl‐CoA. No other propionyl‐CoA carboxylase activity, different from that catalysed by ACC220, could be detected from either reproductive or vegetative organs of pea plants at any stage of development.
Purification and Characterization of Recombinant Human Apolipoprotein A‐II Expressed in Escherichia coli
Authors: Lopez José , Latta Martine , Collet Xavier , Vanloo Berlinda , Jung Gérard , Denefle Patrice , Rosseneu Maryvonne , Chambaz Jean
Published: March 2005

We have expressed recombinant human apolipoprotein A‐II (apoA‐II) in Escherichia coli, as a fusion protein with Schistosoma japonicum glutathione‐S ‐transferase (GST). The GST – AII fusion protein was recovered by affinity chromatography using glutathione as a ligand. After thrombin cleavage and removal of the GST carrier, recombinant apoA‐II was obtained in a highly purified form and was exclusively composed of dimeric apoA‐II. Kinetics of association to dimyristoylglycerophosphocholine (Myr2GroPCho) vesicles showed that recombinant apoA‐II exhibited the same pattern of association as human plasma apoA‐II. Electron microscopic analysis of the complexes showed a typical pattern of rouleaux, characteristic of stacked discs, with a diameter similar to that determined by gradient‐gel electrophoresis. Circular dichroism measurements showed that the α‐helical content of both plasma and recombinant apoA‐II increased similarly when the proteins associated with Myr2GroPCho vesicles, at the expense of a random‐coil structure. Lipid‐bound apoA‐II consisted of 70–72% α helices, suggesting the presence of three 18‐residue α helices/apoA‐II monomer. Cross‐linking experiments indicated that Myr2GroPCho complexes contained two molecules dimeric apoA‐II/vesicle. Recombinant apoA‐II was as efficient as plasma apoA‐II in associating with HDL subclasses, and in displacing apoA‐I from dipalmitoylglycerophosphocholine/cholesterol/apoA‐I complexes, most likely due to its highly ordered secondary structure when associated with Myr2GroPCho vesicles. These findings demonstrate that recombinant apoA‐II exhibits the same structural and functional properties as human plasma apoA‐II. Thus, the expression system utilized is appropriate to produce mutagenized forms to further structure/function analysis.
Structural Characterisation of Human Stefin A in Solution and Implications for Binding to Cysteine Proteinases
Authors: Martin John R. , Jerala Roman , Kroon‐Žitko Louise , Žerovnik Eva , Turk Vito , Waltho Jonathan P.
Published: March 2005

Stefin A is a member of the cystatin superfamily of proteins which are tight and reversibly binding inhibitors of the papain‐like cysteine proteinases. The 1H‐NMR and 15N‐NMR resonances of human stefin A have been sequentially assigned using two‐dimensional homonuclear and hetero‐nuclear NMR techniques in conjunction with three‐dimensional heteronuclear methods. Characteristic sequential and medium range NOE contacts, J constants and hydrogen exchange data have been used to identify the secondary structural elements of the protein which consists of five anti‐parallel β‐strands and a single α‐helix. There is much similarity between the secondary structural features of stefin A and the homologous protein stefin B in its complex with papain [Stubbs, M. T., Laber, B., Bode, W., Huber, R., Jerala, R., Lenarčič, B. & Turk, V. (1990) EMBO. J. 9, 1939–1947] but also some important differences in regions which are fundamental to the binding event. The principal difference is the presence of two conformationally unrestricted regions in stefin A that form two of the components of the tripartite wedge which docks into the active site of the target proteinase. Specifically, these regions are the five N‐terminal residues and the second binding loop, which form a turn and a short helix respectively, in the bound conformation of stefin B.
Structure of the Escherichia coli 024 and 056 O‐Specific Sialic‐Acid‐Containing Polysaccharides and Linkage of these Structures to the Core Region in Lipopolysaccharides
Authors: Gamian Andrzej , Kenne Lennart , Mieszała Małgorzata , Ulrich Jacques , Defaye Jacques
Published: March 2005

The lipopolysaccharides from Escherichia coli O24 and O56 could be separated into higher‐molecular‐mass and lower‐molecular‐mass fractions. Mild acid hydrolysis of lipopolysaccharides of both serotypes released an O‐specific polysaccharide and a tetrasaccharide repeating unit. Oligomers of the repeating unit, the core and the oligosaccharide that contains a fragment of the repeating unit linked to the core region were also obtained according to hydrolysis conditions. On the basis of sugar and methylation analyses, Smith degradation, fast‐atom‐bombardment mass spectrometry and NMR spectroscopy of the hydrolysis products, the biological repeating units of the 0‐specific polysaccharides were shown to be the following tetrasaccharides:

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and

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The structures differ from the structures proposed previously by Kogan et al. [Kogan, G., Shashkov, A. S., Jann, B. & Jann, K. (1993) Carbohydr. Res. 238, 261–270; Kogan, G., Jann, B. & Jann, K. (1993) Carbohydr. Res. 238, 335–338].

The O‐specific repeating unit in E. coli O24 lipopolysaccharide is linked to O6 of the terminal D‐galactose in the core region, whereas in O56 LPS the repeating unit is linked to O4 of a subterminal D‐glucose residue in an R2 type core.
Processing of U14 Small Nucleolar RNA from Three Different Introns of the Mouse 70‐kDa‐Cognate‐Heat‐Shock‐Protein Pre‐Messenger RNA
Authors: Barbhaiya Hemang , Leverette Robert D. , Liu Joyce , Maxwell E. Stuart
Published: March 2005

U14 is a small nucleolar RNA required for the processing of eukaryotic rRNA precusors. The U14 genes of mouse as well as rat, hamster, human, Xenopus and trout are encoded within introns of the constitutively expressed 70‐kDa‐cognate‐heat‐shock protein gene (hsc70). We demonstrate here that U14.6 and U14.8 snRNAs, in addition to the previously characterized U14.5, are processed from their respective introns when hsc70 pre‐mRNA transcripts containing these intronic snRNAs are injected into Xenopus oocyte nuclei. Identical intermediates are observed in the processing of all three mouse U14 snRNAs indicating similar processing pathways. The production of U14 snRNA processing intermediates possessing either mature 5′ or 3′ termini demonstrated that processing can occur at either end independent of maturation at the other terminus. Processing of U14.6 from hsc70 intron 6 is not dependent upon the base pairing of intron sequences flanking the 5′ and 3′ termini of the encoded U14 snRNA molecule. Therefore, excision of an intronic snRNA does not require extending the 5′,3′ terminal helix of U14 snRNA secondary structure into flanking intron regions as originally suggested. Microinjection of the plasmid vector containing the mouse hsc70/U14.5 snRNA coding region revealed that undetermined plasmid sequences can serve as non‐specific promoters to generate spurious RNA transcripts. The processing of these transcripts and examination of the plasmid‐initiated transcriptional‐start sites indicated that a U14‐specific promoter is not present in or around the intron‐encoded U14.5 gene. These results strongly suggest that biosynthesis of mouse U14 snRNA results from an intron‐processing pathway.
Thermodynamics of the Formylmethanofuran Dehydrogenase Reaction in Methanobacterium Thermoautotrophicum
Authors: Bertram Peter A. , Thauer Rudolf K.
Published: March 2005

Purified formylmethanofuran dehydrogenase from Methanobacterium thermoautotrophicum, which is a thermophilic methanogenic Archaeon growing on H2 and CO2, was shown to catalyze the reversible reduction of CO2 to N‐formylmethanofuran with 1,1′,2,2′‐tetramethylviologen (E′o=− 550 mV) as electron donor. The rate of CO2 reduction was approximately 25 times higher than the rate of N‐formylmethanofuran dehydrogenation. From determinations of equilibrium concentrations at 60°C and pH 7.0 a midpoint potential (E′o) for the CO2+ methanofuran/formylmethanofuran couple of approximately – 530 mV was estimated. The initial step of methanogenesis from CO2 thus has a midpoint potential considerably more negative than that of the H+/H2 couple (E′o=−460 mV at 60°C). Evidence is described indicating that the as‐yet unidentified physiological electron donor of the formylmethanofuran dehydrogenase is present in the soluble cell fraction.
Purification and Primary Structure of Metallothioneins Induced by Cadmium in the Protists Tetrahymena Pigmentosa and Tetrahymena Pyriformis
Authors: Piccinni Ester , Staudenmann Werner , Albergoni Vincenzo , De Gabrieli Raffaele , James Peter
Published: March 2005

Tetrahymena pyriformis and Tetrahymena pigmentosa grown in the presence of a non‐toxic dose of cadmium, accumulate the metal in the cytosol. Purification by gel‐permeation, ion‐exchange and reverse‐phase high‐performance liquid chromatography showed that the metal is bound principally to newly formed proteins with ultraviolet spectra and cysteine contents similar to those of Cd2+‐metallothioneins from multicellular organisms. The isolated proteins revealed that the two species of ciliates each express two Cd2+‐isothioneins. The primary structures determined by both Edman degradation and mass spectrometry revealed that the equivalent proteins from T. pyriformis and T. pigmentosa have identical sequences and that the two isoforms in each species differ only by the presence or absence of a lysine residue at the N‐terminus. The development of automated mass spectrometric sequence analysis algorithms combined with an accurate determination of the molecular mass allowed the rapid confirmation of the sequences.

The Tetrahymena metallothionein sequences are unusually long (105 and 104 amino acids) and show a unique internal homology which suggests that the proteins arose by gene duplication. The chains contain 31 cysteine residues, 15 of which are arranged in motifs characteristic of the mammalian metallothioneins; the remaining residues show several unique repeating motifs, which could have interesting consequences for the tertiary structure of the metal‐binding sites. Amino acid sequences of Tetrahymena metallothioneins have some similarity with other eukaryotic metallothioneins. A comparison on the basis of optimised FASTA scores, shows a closer relationship with horse metallothionein‐1B.
Evidence for Domain Structures of the Trifunctional Protein and the Tetrafunctional Protein Acting in Glyoxysomal Fatty Acid β‐Oxidation
Authors: Gühnemann‐Schäfer Kerstin , Engeland Kurt , Linder Dietmar , Kindl Helmut
Published: March 2005

In plant glyoxysomes, an enzyme activity responsible for a particular step in the fatty acid β‐oxidation is located on more than one protein species. Various monofunctional enzymes and two forms of a multifunctional protein are involved in the degradation of cis‐unsaturated fatty acids. Δ3, Δ2‐Enoyl‐CoA isomerase activity, previously found to be located on a monofunctional dimeric protein, is attributable to one form of the monomeric multifunctional protein (MFP). The presence or absence of isomerase activity allows us to differentiate between the tetrafunctional 76.5‐kDa isoform (MFP II) and the trifunctional 74‐kDa isoform (MFP I) in cucumber (Cucumis sativus) cotyledons. Both MFP I and MFP II exhibited blocked N‐terminal structures. MFP I and MFP II are distinguishable from each other by their susceptibility to limited proteolysis. A series of examples is presented describing the preparation of enzymically active proteolytic fragments. We demonstrate that both forms of the monomeric MFP are composed of domains separable from each other without loss of activity. By fragmentation of MFP I and subsequent chromatography, a 60‐kDa peptide was purified retaining hydratase and epimerase activity but lacking dehydrogenase activity. In addition, a highly positively charged fragment was observed carrying solely dehydrogenase activity. From MFP II, a 36‐kDa fragment with hydratase activity was characterized. An enzymically inactive 46‐kDa fragment was prepared from MFP II and sequenced at its unblocked N‐terminus.
Influence of the Four Leader Sequences of the Human Insulin‐Like‐Growth‐Factor‐2 mRNAs on the Expression of Reporter Genes
Authors: Moor Cornelia H. De , Jansen Maarten , Bonte Edgar J. , Thomas Adri A. M. , Sussenbach John S. , Brande J. Leo Van Den
Published: March 2005

The human insulin‐like‐growth‐factor‐2 (IGF‐2) gene generates mRNAs with four different leader sequences, but with identical coding and trailing regions. Previous research has revealed that the leader‐2‐containing and leader‐4‐containing mRNAs are completely polysomal, whereas mRNAs possessing leader‐3 are predominantly present in the untranslated free messenger ribonucleoprotein particle (mRNP), both in cell lines and in foetal liver tissue. To investigate the influence of the IGF‐2 leader sequences on expression of the gene, IGF‐2 leader–luciferase and leader‐chloramphenicol acetyltransferase fusion constructs were transfected transiently into different cell lines. In these experiments, the levels of expression obtained by constructs with leader‐1, leader‐2 and leader‐4 were very similar, both at the level of mRNA and protein. Leader‐3, however, strongly repressed the expression of the fusion mRNA via an unknown mechanism. This repression appeared to be confined to nucleotides at positions 328–906 of the leader sequence. The remaining 5′ part of the leader sequence was efficient both in RNA expression and in translation, but the 3′ part of the leader (nucleotides 906–1180) again moderately repressed luciferase expression, possibly due to endonucleolytic cleavage in this region of the RNA.

To evaluate the effect of the IGF‐2 leaders on in vitro translation, leader–chloramphenicol acetyltransferase fusion mRNAs were synthesized and translated in reticulocyte lysates. Compared to a chloramphenicol acetyltransferase control RNA, leader‐1‐chloramphenicol acetyltransferase mRNA translated over 20‐fold less efficiently, whereas leader‐2 repressed translation of its chloramphenicol acetyltransferase mRNA moderately (3–5 fold). Despite a general improvement of the translation efficiency upon translation in HeLa lysate, these discrepancies with the transfection data persisted. Translation of leader‐3‐containing dNAs in reticulocyte lysates was barely detectable. The whole 5′ region of leader‐3, up to nucleotide 614, could be shown to be repressive. Only leader‐4 directed translation of the chloramphenicol acetyltransferase open reading frame efficiently. As with leader‐1 and leader‐2, this L4‐chloramphenicol acetyltransferase mRNA translated in a cap‐dependent manner under the conditions of our experiments; translation of this mRNA was relatively resistant to addition of cap analogue.

We conclude that all four IGF‐2 leader sequences differ in their translational properties. This makes it likely that changes in the translational machinery will affect the expression of the various IGF‐2 mRNAs differentially.
Facilitative glucose transporters
Authors: MUECKLER Mike
Published: March 2005

Facilitative glucose transport is mediated by members of the Glut protein family that belong to a much larger superfamily of 12 transmembrane segment transporters. Six members of the Glut family have been described thus far. These proteins are expressed in a tissue‐ and cell‐specific manner and exhibit distinct kinetic and regulatory properties that reflect their specific functional roles. Glut1 is a widely expressed isoform that provides many cells with their basal glucose requirement. It also plays a special role in transporting glucose across epithelial and endothelial barrier tissues. Glut2 is a high‐Km isoform expressed in hepatocytes, pancreatic β cells, and the basolateral membranes of intestinal and renal epithelial cells. It acts as a high‐capacity transport system to allow the uninhibited (non‐rate‐limiting) flux of glucose into or out of these cell types. Glut3 is a low‐Km isoform responsible for glucose uptake into neurons. Glut4 is expressed exclusively in the insulin‐sensitive tissues, fat and muscle. It is responsible for increased glucose disposal in these tissues in the postprandial state and is important in whole‐body glucose homeostasis. Glut5 is a fructose transporter that is abundant in spermatozoa and the apical membrane of intestinal cells. Glut7 is the transporter present in the endoplasmic reticulum membrane that allows the flux of free glucose out of the lumen of this organelle after the action of glucose‐6‐phosphatase on glucose 6‐phosphate. This review summarizes recent advances concerning the structure, function, and regulation of the Glut proteins.
Luminescence spectroscopy of pyridoxic acid and pyridoxic acid bound to proteins
Authors: KWON Oh‐Shin , BLAZQUEZ Manuel , CHURCHICH Jorge E.
Published: March 2005

Luminescence techniques, i.e. fluorescence and phosphorescence, have been employed to study pyridoxic acid bound to proteins through a stable amide linkage. Proteins tagged with 4‐pyridoxic acid display the following fluorescence properties: (a) emission and excitation spectra centered at around 430 and 320 nm, respectively; (b) fluorescence quantum yields of 0.3–0.4 and (c) average decay times covering the range 8–9.6 ns. The fluorescence properties of the probe have been used to study the dynamics of the protein in the nanosecond time scale.

In the absence of molecular oxygen, free and bound 4‐pyridoxic acid exhibit long‐lived emission at room temperature. The long‐lived emission is red‐shifted when compared to fluorescence and decays with average life times ranging over 2.2–0.6 ms depending on the nature of the protein.

The fluorophore pyridoxic acid covalently linked to proteins is suitable to study the dynamics of proteins, i.e. fast and slow motions of the macromolecule in the nanosecond and millisecond time scales, respectively.
Conformational study of a salivary proline‐rich protein repeat sequence
Authors: MURRAY Nicola J. , WILLIAMSON Michael P.
Published: March 2005

Salivary proline‐rich proteins have a repetitive primary structure particularly rich in the amino acids proline, glutamine and glycine. One of the biological roles of these proteins is to bind and precipitate polyphenols (vegetable tannins) present in the diet (e.g. tea, coffee, fruit, chocolate) neutralising their harmful actions which include nutritional loss, inhibition of gut enzymes and oesophageal cancer. Two peptides overlapping in sequence, corresponding to the mouse salivary proline‐rich protein MP5 repeat sequence: QGPPPQGGPQQRPPQPGNQ and GPQQRPPQPGNQQGPPPQGGPQ have been synthesised and studied in H2O/(2H6)dimethyl sulphoxide (9:1, by vol.) using 1H‐NMR spectroscopy. Low‐temperature far‐ultraviolet CD spectroscopy and NMR conformational parameters indicate that the peptides adopt an extended random coil conformation in solution. There is no evidence for a defined polyproline type II helix in the peptides, despite the high proline content. NMR data show that the trans‐proline isomer predominates to at least 90%.
Isolation and characterization of hirustasin, an antistasin‐type serine‐proteinase inhibitor from the medical leech Hirudo medicinalis
Authors: SÖLLNER Christa , MENTELE Reinhardt , ECKERSKORN Christoph , FRITZ Hans , SOMMERHOFF Christian P.
Published: March 2005

Antistasin, a potent inhibitor of the blood coagulation factor Xa, is the prototype of a novel family of serine‐proteinase inhibitors. We have now isolated, sequenced and characterized an antistasin‐type inhibitor from the medical leech Hirudo medicinalis. Hirustasin (Hirudo antistasin) was purified to apparent homogeneity by cation‐exchange and affinity chromatography. Amino acid sequencing of the 55 amino acid protein (Mr 5866) revealed that hirustasin is the only antistasin‐type protein known to consist of one domain only; 27% and 32% sequence identity was found to the first and second domains of antistasin, respectively, and a nearly exact conservation of the spacing of the ten cysteine residues. Hirustasin is the first inhibitor of tissue kallikrein identified in leeches, and is also a tight‐binding inhibitor of trypsin, chymotrypsin and neutrophil cathepsin G. However, despite the high similarity to antistasin, particularly in the vicinity of the putative reactivesite peptide bond, hirustasin neither inhibits blood coagulation in vitro nor amidolytic activity of isolated factor Xa. Thus, structural elements other than the reactive site sequence significantly influence the specificity of antistasin‐type proteinase inhibitors.
Probing conformational states of spin‐labeled aspartate aminotransferase by ESR
Authors: STERK Michael , HAUSER Helmut , MARSH Derek , GEHRING Heinz
Published: March 2005

Mitochondrial aspartate aminotransferase was selectively labeled with various maleimide‐linked nitroxide spin labels at the conformationally sensitive Cys166. The mobility of the spin group was found to increase with increasing length of the spacer between the nitroxide and maleimide moiety. The label with the ethylcarbamoyl group, a spacer of intermediate length, responded sensitively to conformational changes of aspartate aminotransferase. The modification with this label decreased the enzymic activity to 30% of its initial value and increased the affinity for various substrates and inhibitors 5 – 10‐fold. Identical ESR spectra were obtained for the pyridoxal and pyridoxamine form of the enzyme. These spectra are complex, consisting of an isotropic and at least two anisotropic components. The spectral complexity is attributed to different modes of interaction of the spin label with its local protein environment giving rise to different motional states. The same changes in the ESR spectra have been observed upon formation of the adsorption complex of the pyridoxal form with a competitive inhibitor and on formation of covalent intermediates of the transamination reaction. Essentially, the isotropic component is converted to a new anisotropic one as the local environment changes due to a conformational adaptation of aspartate aminotransferase. The ESR data are consistent with an equilibrium between two conformational states of the enzyme but inconsistent with individual protein conformations of the various intermediates of the transamination reaction. The two conformational states may be assigned to the open and closed conformations as defined by X‐ray crystallography. In the adsorption complex of the pyridoxal enzyme, and in the covalent intermediates, the two‐state equilibrium appears to be shifted towards the closed conformation in which the spin label is more rigidly bound, as also suggested by molecular dynamic simulations of the label modelled into aspartate aminotransferase. In contrast the formation of adsorption complexes between the pyridoxamine form and aspartate or maleate was not accompanied by the same shift of the conformational equilibrium.
Kinetic resolution of the reaction catalysed by proton‐translocating transhydrogenase from Escherichia coli as revealed by experiments with analogues of the nucleotide substrates
Authors: HUTTON Mike , DAY Joanna M. , BIZOUARN Tania , JACKSON J. Baz
Published: March 2005

The mechanism, by which transhydrogenase couples transfer of H− equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane, has been investigated in the solubilised, purified enzyme from Escherichia coli using analogues of the nucleotide substrates. The key observation was that, at low pH and ionic strength, solubilised transhydrogenase catalysed the very rapid reduction of acetylpyridine adenine dinucleotide (an analogue of NAD+) by NADH, but only in the presence of either NADP+ or NADPH. This indicates that the rates of release of NADP+ and NADPH from their binary complexes with the enzyme are slow. The dependences on pH and salt concentration suggest that (a) release of both NADP+ and NADPH are accompanied by the release of H+ from the enzyme and (b) increased ionic strength decreases the value of the pKa of the group responsible for H+ release. Modification of the enzyme with N,N1‐dicyclohexylcarbodiimide led to inhibition of the rate of release of NADP+ and NADPH from the enzyme, but had a much smaller effect on the binding and release of NAD+, NADH and their analogues and on the interconversion of the ternary complexes of the enzyme with its substrates.

It is considered that the binding and release of H+, which accompany the binding and release of NADP+/NADPH, might be central to the mechanism of proton translocation by the enzyme in its membrane‐bound state.
Purification, characterization, primary structure, crystallization and preliminary crystallographic study of a serine proteinase from Streptomyces fradiae ATCC 14544
Authors: KITADOKORO Kengo , TSUZUKI Hiroshige , NAKAMURA Etsuo , SATO Tomohiro , TERAOKA Hiroshi
Published: March 2005

A proteinase having wide substrate specificity was isolated from Streptomyces fradiae ATCC 14544. This proteinase, which we propose to call SFase‐2, was purified from the culture filtrate by S‐Sepharose chromatography. The purified enzyme showed an apparent molecular mass of 19 kDa on SDS/PAGE. When synthetic peptides were used as substrates, SFase‐2 showed broad substrate specificity. It also hydrolyzed keratin, elastin and collagen as proteinaceous substrates. It was completely inhibited by diisopropylfluorophosphate and chymostatin, but not by tosylphenyl‐alaninechloromethane, tosyllysinechloromethane or EDTA, indicating that it can be classified as a serine proteinase.

The matured protein sequence of SFase‐2 was determined by a combination of amino acid sequencing and the DNA sequencing of the gene. SFase‐2, consisting of 191 amino acids, is a novel proteinase. It showed 76% similarity in the amino acid sequence with Streptomyces griseus proteinase A [Johnson P. and Smillie L. B. (1974) FEBS Lett. 47, 1–6].

For insight into the three‐dimensional structure of SFase‐2, we obtained single crystals by the vapor diffusion method using sodium phosphate as a precipitant. These crystals belonged to the orthorhombic, space group P212121 with cell dimensions a= 6.92 nm, b= 7.28 nm, c= 2.99 nm; one molecule was present in the asymmetric unit.
A common response element mediates differential effects of phorbol esters and forskolin on type‐1 plasminogen activator inhibitor gene expression in human breast carcinoma cells
Authors: KNUDSEN Helle , OLESEN Tina , RICCIO Andrea , UNGARO Paola , CHRISTENSEN Lise , ANDREASEN Peter A.
Published: March 2005

We have characterized regulation of type‐1 plasminogen activator inhibitor (PAI‐1) gene expression by phorbol 12‐myristate 13‐acetate (PMA) and the cAMP‐inducing agent forskolin in the human breast carcinoma cell line MCF‐7. PMA caused a strong induction of PAI‐1, while forskolin suppressed the PMA response. Transfection experiments with fusion genes showed that sequences mediating PMA induction as well as forskolin suppression were present between base pairs –100 and –30 of the 57prime;‐flanking region of the PAI‐1 gene. The region was found to contain two Sp1 binding sites. A proximal sequence in the region, TGAGTTCA (P box), with sequence similarity to phorbol ester response elements (TRE) as well as to cAMP response elements (CRE), bound a low‐abundance, as yet unidentified nuclear protein in MCF‐7 cells. This sequence had a higher affinity to purified c‐jun homodimer than to c‐jun/c‐fos heterodimer in MCF‐7 nuclear extracts; it had no affinity to the proteins binding to CRE consensus sequences in these extracts. A distal TRE‐like sequence, TGAGTGG (D box), had a weak affinity to c‐jun/c‐fos heterodimer and c‐jun homodimer; binding of proteins to this sequence was facilitated by binding of proteins to the P box. Both the P box and the D box were necessary for PMA responsiveness, suggesting a cooperativity between the two binding sites. A mutation of the P box removing the CRE similarity abolished the forskolin suppression of the PMA response. We propose that the protein kinase C and the protein kinase A signal‐transduction pathways, with opposite effects on PAI‐1 gene expression, converge by modulating differently P‐box‐binding proteins.