bluuebunny/crossref_metadata_2025 huggingface dataset:
A treatment trade‐off based decision aid for patients with locally advanced non‐small cell lung cancer
Authors: Brundage Michael D. , Feldman‐Stewart Deb , Dixon Peter , Gregg Richard , Youssef Youssef , Davies Diane , Mackillop William J.
Published: March 2003

Purpose To describe the structure and use of a decision aid for patients with locally advanced non‐small cell lung cancer (LA‐NSCLC) who are eligible for combined‐modality treatment (CMT) or for radiotherapy alone (RT).

Methods The aid included a structured description of the treatment options and trade‐off exercises designed to help clarify the patient’s values for the relevant outcomes by determining the patient’s survival advantage threshold (the increase in survival conferred by CMT over RT that the patient deemed necessary for choosing CMT). Additional outcome measures included each patient’s strength of treatment preference, decisional conflict, objective understanding of survival information, decisional role preference, and evaluation of the aid itself.

Results Twenty‐five patients met the eligibility criteria for study. Of these, seven declined the decision aid because they had a clear treatment preference (four chose CMT and three chose RT). The remaining 18 participants completed the decision aid; 16 chose CMT and two chose RT. All 18 patients wished to participate in the decision to some extent. All patients reported that using the decision support was useful to them and recommended its use for others. No patient or physician reported that the aid interfered with the physician‐patient relationship. Patients’ 3‐year survival advantage thresholds, and their median survival advantage thresholds, were each strongly correlated with their strengths of treatment preference (ρ=0.80,P < 0.001 andρ=0.77,P < 0.001, respectively). For all but one patient, either their 3‐year or median survival threshold was consistent with their final treatment choice. Eight patients reported a stronger treatment preference after using the decision aid.

Conclusions We conclude that a treatment trade‐off based decision aid for patients with locally advanced non‐small cell lung cancer is feasible, that it demonstrates internal consistency and convergent validity, and that it is favourably evaluated by patients and their physicians. The aid seems to help patients understand the benefits and risks of treatment and to choose the treatment that is most consistent with their values.
Patients' attitudes to medicines and expectations for prescriptions
Authors: Britten Nicky , Ukoumunne Obioha C. , Boulton Mary G.
Published: March 2003

Background  Recent research has shown that patients' expectations for prescriptions influence doctors' prescribing decisions, but little is known of the antecedents of these expectations.

Objectives  To test earlier qualitative research about patients' views of medicines; to describe the demographic characteristics of those holding orthodox and unorthodox views of medicines; to investigate the relationship between patients' ideal and predicted expectations for prescriptions; and to determine the relative effects of attitudinal, demographic, organizational and illness variables on these expectations.

Design  Questionnaire survey of patients consulting general practitioners.

Setting and participants  A total of 544 patients and 15 doctors in four general practices.

Main variables studied  Patients' attitudes to medicines; patients' demographic characteristics; organizational variables; aspects of patients' presenting problems.

Outcome measures  Patients' ideal and predicted expectations for prescriptions.

Results  Orthodox and unorthodox attitudes to medicines can be measured quantitatively, and ethnicity was the only demographic variable associated with both. Ideal and predicted expectations for prescriptions were closely related to each other but differed in their antecedents. Both types of expectations were associated with attitudinal, demographic, organizational and illness variables. Ideal expectations were influenced by orthodox and unorthodox attitudes to medicines, while predicted expectations were only influenced by orthodox attitudes.

Conclusions  Future studies of patients' expectations for health services should distinguish between ideal and predicted expectations, and should consider the range of possible influences on these expectations. In particular, the effect of the organization and context of health services should be investigated.
Concentration‐dependent reversible activation‐inhibition of human butyrylcholinesterase by tetraethylammonium ion
Authors: Stojan Jure ,   Marko Goličnik, Froment Marie‐Thérèse , Estour Francois , Masson Patrick
Published: March 2003

Tetraalkylammonium (TAA) salts are well known reversible inhibitors of cholinesterases. However, at concentrations around 10 mm, they have been found to activate the hydrolysis of positively charged substrates, catalyzed by wild‐type human butyrylcholinesterase (EC 3.1.1.8) [Erdoes, E.G., Foldes, F.F., Zsigmond, E.K., Baart, N. & Zwartz, J.A. (1958) Science128, 92]. The present study was undertaken to determine whether the peripheral anionic site (PAS) of human BuChE (Y332, D70) and/or the catalytic substrate binding site (CS) (W82, A328) are involved in this phenomenon. For this purpose, the kinetics of butyrylthiocholine (BTC) hydrolysis by wild‐type human BuChE, by selected mutants and by horse BuChE was carried out at 25 °C and pH 7.0 in the presence of tetraethylammonium (TEA). It appears that human enzymes with more intact structure of the PAS show more prominent activation phenomenon. The following explanation has been put forward: TEA competes with the substrate at the peripheral site thus inhibiting the substrate hydrolysis at the CS. As the inhibition by TEA is less effective than the substrate inhibition itself, it mimics activation. At the concentrations around 40 mm, well within the range of TEA competition at both substrate binding sites, it lowers the activity of all tested enzymes.
Interallelic recombination is probably responsible for the occurrence of a new αs1‐casein variant found in the goat species
Authors: Bevilacqua Claudia , Ferranti Pasquale , Garro Giuseppina , Veltri Cristina , Lagonigro Raffaella , Leroux Christine , Pietrolà Emilio , Addeo Francesco , Pilla Fabio , Chianese Lina , Martin Patrice
Published: March 2003

The αs1‐casein (αs1‐Cas) locus in the goat is characterized by a polymorphism, the main feature of which is to be qualitative as well as quantitative. A systematic analysis performed in an autochthon southern Italy breed identified a new rare allele (M), which was characterized at both the protein and genomic level. The M protein displays the slowest electrophoretic mobility of the αs1‐Cas variants described so far. MS and automated Edman degradation experiments showed that this behavior was due to the loss of two phosphate residues in the multiple phosphorylation site (64SP‐SP‐SP‐SP‐SP‐E‐70E) consecutively to a Ser→Leu substitution at position 66 of the peptide chain (64S‐SP‐L‐SP‐SP‐E‐70E). This was confirmed by sequencing a genomic DNA fragment encompassing exon 9 where the 8th codon (TCG) was shown to be mutated to TTG. Sequencing of amplified genomic DNA segments spanning the 5′ and 3′ flanking regions of each exon allowed us to identify 23 single nucleotide polymorphisms and two insertion/deletion events in the coding as well as the noncoding regions. A comparison of specific haplotypes defined for each of the αs1‐CasF, A and M␣alleles indicates that the M allele probably arises from interallelic recombination between alleles A and B2, followed by a C→T transition at nucleotide 23 of the ninth exon. The region encompassing the recombination break point was putatively located between nucleotide 86 upstream and nucleotide 40 downstream of exon 8. Interallelic recombination therefore appears to be a possible means of generating allelic diversity at the αs1‐Cas locus, at least in the goat. The previously proposed molecular phylogeny must now be revised, possibly starting from two ancestral allelic lineages.
NMR‐based determination of the binding epitope and conformational analysis of MUC‐1 glycopeptides and peptides bound to the breast cancer‐selective monoclonal antibody SM3
Authors: Möller Heiko , Serttas Nida , Paulsen Hans , Burchell Joy M. , Taylor‐Papadimitriou Joyce ,   Bernd Meyer
Published: March 2003

Mucin glycoproteins on breast cancer cells carry shortened carbohydrate chains. These partially deglycosylated mucin 1 (MUC‐1) structures are recognized by the monoclonal antibody SM3, which is being tested for its diagnostic utility. We used NMR spectroscopy to analyze the binding mode and the binding epitope of peptide and glycopeptide antigens to the SM3 antibody. The pentapeptide PDTRP and the glycopentapeptide PDT(O‐α‐d‐GalNAc)RP are known ligands of the monoclonal antibody. The 3D structures of the ligands in the bound conformation were determined by analyzing trNOESY build‐up rates. The peptide was found to adopt an extended conformation that fits into the binding pocket of the antibody. The binding epitopes of the ligands were determined by saturation transfer difference (STD) NMR spectroscopy. The peptide's epitope is predominantly located in the N‐terminal PDT segment whereas the C‐terminal RP segment has fewer interactions with the protein. In␣contrast, the glycopeptide is interacting with SM3 utilizing all its amino acids. Pro1 shows the strongest binding effect that slightly decays towards Pro5. The GalNAc residue interacts mainly via the N‐acetyl residue while the other protons show less interactions similar to that of Pro5. The glycopeptide in the bound state also has an extended conformation of the peptide with the carbohydrate oriented towards the N‐terminus. Docking studies showed that peptide and glycopeptide fit the binding pocket of the mAb SM3 very well.
Lectin–sugar interaction
Authors: Neumann Dirk , Kohlbacher Oliver , Lenhof Hans‐Peter , Lehr Claus‐Michael
Published: March 2003

Although a steadily increasing number of protein–ligand docking experiments have been performed successfully, there are only few studies concerning protein–sugar interactions. In this study, we investigate the interaction of wheat germ agglutinin (WGA) with N‐acetylglucosamine and a number of its derivatives and predict the binding free energies using flexible docking techniques. To assess the quality of our predictions, we also determined those binding free energies experimentally in cell‐binding studies. The predicted binding site, ligand orientation, and details of the binding mode are in perfect agreement with the known crystal structure of WGA with a sialoglycopeptide. Furthermore, we obtained an excellent linear correlation of our predicted binding free energies with both our own data and experimental data from the literature [Monsigny, M., Roche, A.C., Sene, C., Maget Dana, R. & Delmotte, F. (1980) Eur. J. Biochem.104, 147–153.]. In both cases, predicted energies were within 1.0 kJ·mol−1 of the experimental value. These results illustrate the usefulness of docking‐based methods for the qualitative and quantitative prediction of protein–carbohydrate interactions. The insights gained from such theoretical studies may be used to complement the results from the still scarce crystal structures.
Purification and biochemical characterization of some of the properties of recombinant human kynureninase
Authors: Walsh Harold A. , Botting Nigel P.
Published: March 2003

Recombinant human kynureninase (l‐kynurenine hydrolase, EC 3.7.1.3) was purified to homogeneity (60‐fold) from Spodoptera frugiperda (Sf9) cells infected with baculovirus containing the kynureninase gene. The purification protocol comprised ammonium sulfate precipitation and several chromatographic steps, including DEAE–Sepharose CL‐6B, hydroxyapatite, strong anionic and cationic separations. The purity of the enzyme was determined by SDS/PAGE, and the molecular mass verified by MALDI‐TOF MS. The monomeric molecular mass of 52.4 kDa determined was > 99.99% of the predicted molecular mass. A UV absorption spectrum of the holoenzyme resulted in a peak at 432 nm. The optimum pH was 8.25 and the enzyme displayed a strong dependence on the ionic strength of the buffer for optimum activity. This cloned enzyme was highly specific for 3‐hydroxykynurenine (Km = 3.0 µm ± 0.10) and was inhibited by l‐kynurenine (Ki = 20 µm), d‐kynurenine (Ki = 12 µm) and a synthetic substrate analogue d,l‐3,7‐dihydroxydesaminokynurenine (Ki = 100 nm). The activity/concentration profile for kynureninase from this source was sigmoidal in all instances. There appeared to be partial inhibition by substrate, and excess pyridoxal 5′‐phosphate was found to be inhibitory.
Mitochondrial DNA deletion mutations
Authors: McKenzie Debbie , Bua Entela , McKiernan Susan , Cao Zhengjin ,   Jonathan Wanagat, Aiken Judd M.
Published: March 2003

Mitochondrial DNA (mtDNA) deletion mutations accumulate with age in tissues of a variety of species. Although the relatively low calculated abundance of these deletion mutations in whole tissue homogenates led some investigators to suggest that these mutations do not have any physiological impact, their focal and segmental accumulation suggests that they can, and do, accumulate to levels sufficient to affect the metabolism of a tissue. This phenomenon is most clearly demonstrated in skeletal muscle, where the accumulation of mtDNA deletion mutations remove critical subunits that encode for the electron transport system (ETS). In this review, we detail and provide evidence for a molecular basis of muscle fiber loss with age. Our data suggest that the mtDNA deletion mutations, which are generated in tissues with age, cause muscle fiber loss. Within a fiber, the process begins with a mtDNA replication error, an error that results in a loss of 25–80% of the mitochondrial genome. This smaller genome is replicated and, through a process not well understood, eventually comprises the majority of mtDNA within the small affected region of the muscle fiber. The preponderance of the smaller genomes results in a dysfunctional ETS in the affected area. As a consequence of both the decline in energy production and the increase in oxidative damage in the region, the fiber is no longer capable of self‐maintenance, resulting in the observed intrafiber atrophy and fiber breakage. We are therefore proposing that a process contained within a very small region of a muscle fiber can result in breakage and loss of muscle fiber from the tissue.
Excessive vitamin A toxicity in mice genetically deficient in either alcohol dehydrogenase Adh1 or Adh3
Authors: Molotkov Andrei , Fan Xiaohong , Duester Gregg
Published: March 2003

Alcohol dehydrogenase (ADH) deficiency results in decreased retinol utilization, but it is unclear what physiological roles the several known ADHs play in retinoid signaling. Here, Adh1, Adh3, and Adh4 null mutant mice have been examined following acute and chronic vitamin A excess. Following an acute dose of retinol (50 mg·kg−1), metabolism of retinol to retinoic acid in liver was reduced 10‐fold in Adh1 mutants and 3.8‐fold in Adh3 mutants, but was not significantly reduced in Adh4 mutants. Acute retinol toxicity, assessed by determination of the LD50 value, was greatly increased in Adh1 mutants and moderately increased in Adh3 mutants, but only a minor effect was observed in Adh4 mutants. When mice were propagated for one generation on a retinol‐supplemented diet containing 10‐fold higher vitamin A than normal, Adh3 and Adh4 mutants had essentially the same postnatal survival to adulthood as wild‐type (92–95%), but only 36% of Adh1 mutants survived to adulthood with the remainder dying by postnatal day 3. Adh1 mutants surviving to adulthood on the retinol‐ supplemented diet had elevated serum retinol signifying a clearance defect and elevated aspartate aminotransferase indicative of increased liver damage. These findings indicate that ADH1 functions as the primary enzyme responsible for efficient oxidative clearance of excess retinol, thus providing protection and increased survival during vitamin A toxicity. ADH3 plays a secondary role. Our results also show that retinoic acid is not the toxic moiety during vitamin A excess, as Adh1 mutants have less retinoic acid production while experiencing increased toxicity.
Importin α binds to an unusual bipartite nuclear localization signal in the heterogeneous ribonucleoprotein type I
Authors: Romanelli Maria G. , Morandi Carlo
Published: March 2003

The heterogeneous nuclear ribonucleoprotein (hnRNP) type I, a modulator of alternative splicing, localizes in the nucleoplasm of mammalian cells and in a discrete perinucleolar structure. HnRNP I contains a novel type of bipartite nuclear localization signal (NLS) at the N‐terminus of the protein that we have previously named nuclear determinant localization type I (NLD‐I). Recently, a neural counterpart of hnRNP I has been identified that contains a putative NLS with two strings of basic amino acids separated by a spacer of 30 residues. In the present study we show that the neural hnRNP I NLS is necessary and sufficient for nuclear localization and represents a variant of the novel bipartite NLS present in the NLD‐I domain. Furthermore, we demonstrate that the NLD‐I is transported into the nucleus by cytoplasmic factor(s) with active transport modality. Binding assays using recombinant importin α show an interaction with NLD‐I similar to that of SV40 large T antigen NLS. Deletion analysis indicates that both stretches of basic residues are necessary for binding to importin α. The above experimental results lead to the conclusion that importin α acts as cytoplasmic receptor for proteins characterized by a bipartite NLS signal that extends up to 37 residues.
Tryptophan fluorescence study of the interaction of penetratin peptides with model membranes
Authors: Christiaens Bart , Symoens Sofie , Vanderheyden Stefan , Engelborghs Yves , Joliot Alain , Prochiantz Alain , Vandekerckhove Joël , Rosseneu Maryvonne , Vanloo Berlinda
Published: March 2003

Penetratin is a 16‐amino‐acid peptide, derived from the homeodomain of antennapedia, a Drosophila transcription factor, which can be used as a vector for the intracellular delivery of peptides or oligonucleotides. To study the relative importance of the Trp residues in the wild‐type penetratin peptide (RQIKIWFQNRRMKWKK) two analogues, the W48F (RQIKIFFQNRRMKWKK) and the W56F (RQI KIWFQNRRMKFKK) variant peptides were synthesized. Binding of the three peptide variants to different lipid vesicles was investigated by fluorescence. Intrinsic Trp fluorescence emission showed a decrease in quantum yield and a blue shift of the maximal emission wavelength upon interaction of the peptides with negatively charged phosphatidylserine, while no changes were recorded with neutral phosphatidylcholine vesicles. Upon binding to phosphatidylcholine vesicles containing 20% (w/w) phosphatidylserine the fluorescence blue shift induced by the W56F‐penetratin variant was larger than for the W48F‐penetratin. Incorporation of cholesterol into the negatively charged lipid bilayer significantly decreased the binding affinity of the peptides. The Trp mean lifetime of the three peptides decreased upon binding to negatively charged phospholipids, and the Trp residues were shielded from acrylamide and iodide quenching. CD measurements indicated that the peptides are random in buffer, and become α helical upon association with negatively charged mixed phosphatidylcholine/phosphatidylserine vesicles, but not with phosphatidylcholine vesicles. These data show that wild‐type penetratin and the two analogues interact with negatively charged phospholipids, and that this is accompanied by a conformational change from random to α helical structure, and a deeper insertion of W48 compared to W56, into the lipid bilayer.
Identification and characterization of the Escherichia coli stress protein UP12, a putative in vivo substrate of GroEL
Authors: Bochkareva Elena S. , Girshovich Alexander S. , Bibi Eitan
Published: March 2003

Many groups of proteins play important roles in the cell's response to various stresses. The molecular chaperone GroEL of Escherichia coli represents one such highly conserved family of stress proteins. We have observed that isolated GroEL complexes from stationary cultures contain various polypeptides that can be released from the chaperonin by GroES and/or ATP, and identified two such polypeptides as the proteins GatY and UP12. Whereas GatY had been isolated previously, as an in vivo substrate of GroEL, the isolation of UP12 in a complex with GroEL was intriguing, because based on sequence similarity it was suggested that UP12 might also be a functional stress protein. UP12 belongs to a family of universal stress proteins (UspA family), of which UspA itself, and three additional paralogues, have been characterized previously. Here we show that UP12 accumulates under various growth inhibitory conditions and induced by heat shock. Furthermore, unlike wild‐type cells, a UP12 deletion mutant recovers slowly from late stationary growth conditions, and has a marked sensitivity to the toxic agent carbonyl cyanide m‐chlorophenyl hydrazone (CCCP). Finally, coimmunoprecipitation experiments confirmed the initial observation that UP12 interacts with GroEL. Therefore, we suggest that UP12 may function as a universal stress protein, interaction of which with GroEL possibly ensures its proper folding state.
Functional integration of mitochondrial and hydrogenosomal ADP/ATP carriers in the Escherichia coli membrane reveals different biochemical characteristics for plants, mammals and anaerobic chytrids
Authors: Haferkamp Ilka , Hackstein Johannes H. P. , Voncken Frank G. J. , Schmit Guillaume , Tjaden Joachim
Published: March 2003

The expression of mitochondrial and hydrogenosomal ADP/ATP carriers (AACs) from plants, rat and the anaerobic chytridiomycete fungus Neocallimastix spec. L2 in Escherichia coli allows a functional integration of the recombinant proteins into the bacterial cytoplasmic membrane. For AAC1 and AAC2 from rat, apparent Km values of about 40 µm for ADP, and 105 µm or 140 µm, respectively, for ATP have been determined, similar to the data reported for isolated rat mitochondria. The apparent Km for ATP decreased up to 10‐fold in the presence of the protonophore m‐chlorocarbonylcyanide phenylhydrazone (CCCP). The hydrogenosomal AAC isolated from the chytrid fungus Neocallimastix spec. L2 exhibited the same characteristics, but the affinities for ADP (165 µm) and ATP (2.33 mm) were significantly lower. Notably, AAC1‐3 from Arabidopsis thaliana and AAC1 from Solanum tuberosum (potato) showed significantly higher external affinities for both nucleotides (10–22 µm); they were only slightly influenced by CCCP.

Studies on intact plant mitochondria confirmed these observations. Back exchange experiments with preloaded E. coli cells expressing AACs indicate a preferential export of ATP for all AACs tested. This is the first report of a functional integration of proteins belonging to the mitochondrial carrier family (MCF) into a bacterial cytoplasmic membrane. The technique described here provides a relatively simple and highly reproducible method for functional studies of individual mitochondrial‐type carrier proteins from organisms that do not allow the application of sophisticated genetic techniques.
The evolution of monomeric and oligomeric βγ‐type crystallins
Authors: D'Alessio Giuseppe
Published: March 2003

The case of homologous monomeric γ‐type and oligomeric β‐type crystallins has been described and analyzed in evolutionary terms. Data and hypotheses from molecular genetics and structural investigations converge and suggest a novel three‐phase model for the evolutionary history of crystallin‐type proteins. In the divergent cascades of monomeric and oligomeric crystallins, a pivotal role was played by alterations in the gene segments encoding the C‐terminal extensions and the intermotif or interdomain linker peptides. These were genomic hot spots where evolution experimented to produce the modern variety of βγ‐crystallin‐type quaternary structures.
Herbaspirillum seropedicae signal transduction protein PII is structurally similar to the enteric GlnK
Authors: Machado Benelli Elaine , Buck Martin , Polikarpov Igor , Maltempi de Souza Emanuel , Cruz Leonardo M. , Pedrosa Fábio O.
Published: March 2003

PII‐like proteins are signal transduction proteins found in bacteria, archaea and eukaryotes. They mediate a variety of cellular responses. A second PII‐like protein, called GlnK, has been found in several organisms. In the diazotroph Herbaspirillum seropedicae, PII protein is involved in sensing nitrogen levels and controlling nitrogen fixation genes. In this work, the crystal structure of the unliganded H. seropedicae PII was solved by X‐ray diffraction. H. seropedicae PII has a Gly residue, Gly108 preceding Pro109 and the main‐chain forms a β turn. The glycine at position 108 allows a bend in the C‐terminal main‐chain, thereby modifying the surface of the cleft between monomers and potentially changing function. The structure suggests that the C‐terminal region of PII proteins may be involved in specificity of function, and nonenteric diazotrophs are found to have the C‐terminal consensus XGXDAX(107–112). We are also proposing binding sites for ATP and 2‐oxoglutarate based on the structural alignment of PII with PII‐ATP/GlnK‐ATP, 5‐carboxymethyl‐2‐hydroxymuconate isomerase and 4‐oxalocrotonate tautomerase bound to the inhibitor 2‐oxo‐3‐pentynoate.
Barrier passage and protein dynamics in enzymatically catalyzed reactions
Authors: Antoniou Dimitri , Caratzoulas Stavros , Kalyanaraman C. , S. Mincer Joshua , Schwartz Steven D.
Published: March 2003

This review describes studies of particular enzymatically catalyzed reactions to investigate the possibility that catalysis is mediated by protein dynamics. That is, evolution has crafted the protein backbone of the enzyme to direct vibrations in such a fashion to speed reaction. The review presents the theoretical approach we have used to investigate this problem, but it is designed for the nonspecialist. The results show that in alcohol dehydrogenase, dynamic protein motion is in fact strongly coupled to chemical reaction in such a way as to promote catalysis. This result is in concert with both experimental data and interpretations for this and other enzyme systems studied in the laboratories of the two other investigators who have published reviews in this issue.
Ruk is ubiquitinated but not degraded by the proteasome
Authors: Verdier Frédérique , Valovka Taras , Zhyvoloup Alexander , Drobot Ludmila B. , Buchman Vladimir , Waterfield Mike , Gout Ivan
Published: March 2003

The regulator of ubiquitous kinase (Ruk) protein, also known as CIN85 or SETA, is an adaptor‐type protein belonging to the CD2AP/CMS family. It was found in complexes with many signaling proteins, including phosphoinositol (PtdIns) 3‐kinase (EC 2.7.1.137), Cbl, GRB2, p130Cas and Crk. Functional analysis of these interactions, implicated Ruk in the regulation of apoptosis, receptor endocytosis and cytoskeletal rearrangements. We have recently demonstrated that overexpression of Ruk induces apoptotic death in neurons, which could be reversed by activated forms of PtdIns 3‐kinase and PKB/Akt. Furthermore, Ruk was shown to be a negative regulator of PtdIns 3‐kinase activity through binding to its P85 regulatory subunit [Gout, I., Middleton, G., Adu, J., Ninkina, N. N., Drobot, L. B., Filonenko, V., Matsuka, G., Davies, A.M., Waterfield, M. & Buchman, V. L. (2000) Embo J.19, 4015–4025]. Here, we report for the first time, that all three isoforms of Ruk (L, M and S) are ubiquitinated. Specific interaction between the E3 ubiquitin ligase Cbl and all three Ruk isoforms was demonstrated by coexpression studies in Hek293 cells. The interaction of Ruk M and S isoforms with Cbl was found to be mediated via heterodimerization with Ruk L. The use of proteosomal and lysosomal inhibitors clearly indicated that ubiquitination of Ruk L does not lead to its degradation. Based on this study, we propose a possible mechanism for the regulation of Ruk function by ubiquitination.
Rum1, an inhibitor of cyclin‐dependent kinase in fission yeast, is negatively regulated by mitogen‐activated protein kinase‐mediated phosphorylation at Ser and Thr residues
Authors: Matsuoka Kentaro , Kiyokawa Nobutaka , Taguchi Tomoko , Matsui Jun , Suzuki Toyo , Mimori Kenichi , Nakajima Hideki , Takenouchi Hisami , Weiran Tang , Katagiri Yohko U. , Fujimoto Junichiro
Published: March 2003

The p25rum1 is an inhibitor of Cdc2 kinase expressed in fission yeast and plays an important role in cell‐cycle control. As its amino‐acid sequence suggests that p25rum1 has putative phosphorylation sites for mitogen‐activated protein kinase (MAPK), we investigated the ability of MAPK to phosphorylate p25rum1. Direct in vitro kinase assay using GST‐fusion proteins of wild‐type as well as various mutants of p25rum1 demonstrated that MAPK phosphorylates the N‐terminal portion of p25rum1 and residues Thr13 and Ser19 are major phosphorylation sites for MAPK. In addition, phosphorylation of p25rum1 by MAPK revealed markedly reduced Cdc2 kinase inhibitor ability of the protein. Together with the fact that replacement of both Thr13 and Ser19 with Glu, which mimics the phosphorylated state of these residues, also significantly reduces the activity of p25rum1 as a Cdc2 inhibitor, it was suggested that the phosphorylation of Thr13 and Ser19 negatively regulates the function of p25rum1. Further evidence indicates that phosphorylation of Thr13 and Ser19 may retain a negative effect on the function of p25rum1 even in vivo. Therefore, MAPK may regulate the function of p25rum1 via phosphorylation of its Thr and Ser residues and thus participate in cell cycle control in fission yeast.
Engineering and mechanistic studies of theArabidopsisFAE1 β‐ketoacyl‐CoA synthase, FAE1 KCS
Authors: Ghanevati Mahin , Jaworski Jan G.
Published: March 2003

TheArabidopsisFAE1 β‐ketoacyl‐CoA synthase (FAE1 KCS) catalyzes the condensation of malonyl‐CoA with long‐chain acyl‐CoAs. Sequence analysis of FAE1 KCS predicted that this condensing enzyme is anchored to a membrane by two adjacent N‐terminal membrane‐spanning domains. In order to characterize the FAE1 KCS and analyze its mechanism, FAE1 KCS and its mutants were engineered with a His6‐tag at their N‐terminus, and expressed inSaccharomyces cerevisiae.The membrane‐bound enzyme was then solubilized and purified to near homogeneity on a metal affinity column. Wild‐type recombinant FAE1 KCS was active with several acyl‐CoA substrates, with highest activity towards saturated and monounsaturated C16 and C18. In the absence of an acyl‐CoA substrate, FAE1 KCS was unable to carry out decarboxylation of [3–14C]malonyl‐CoA, indicating that it requires binding of the acyl‐CoA for decarboxylation activity. Site‐directed mutagenesis was carried out on the FAE1 KCS to assess if this condensing enzyme was mechanistically related to the well characterized soluble condensing enzymes of fatty acid and flavonoid syntheses. A C223A mutant enzyme lacking the acylation site was unable to carry out decarboxylation of malonyl‐CoA even when 18:1‐CoA was present. Mutational analyses of the conserved Asn424 and His391 residues indicated the importance of these residues for FAE1‐KCS activity. The results presented here provide the initial analysis of the reaction mechanism for a membrane‐bound condensing enzyme from any source and provide evidence for a mechanism similar to the soluble condensing enzymes.
Antiproliferative proteins of the BTG/Tob family are degraded by the ubiquitin‐proteasome system
Authors: Sasajima Hitoshi , Nakagawa Koji , Yokosawa Hideyoshi
Published: March 2003

BTG/Tob family proteins, which are characterized by similarities in their N‐terminal BTG/Tob homology domains, control cell growth negatively. Among the BTG/Tob family members, BTG2/TIS21/PC3 proteins have been reported to have short lives and to be degraded by the proteasome. However, the mechanisms regulating the stabilities of other BTG/Tob family proteins have not yet been clarified. Here, we report that BTG1, Tob, and Tob2 proteins, as well as BTG2 protein, are degraded by the ubiquitin–proteasome system; the degradation of Tob protein in HeLa cells and the degradation of BTG1, BTG2, Tob and Tob2 proteins transiently expressed in HEK293 cells were inhibited by treatments with proteasome‐specific inhibitors. Co‐expression of BTG1, BTG2, Tob, or Tob2 protein with ubiquitin in HEK293 cells revealed specific multiubiquitination of each of the four proteins. Although the full‐length and N‐terminal truncated forms of BTG1, BTG2, Tob, and Tob2 proteins were unstable, the respective C‐terminal truncated forms were found to be almost stable, suggesting that the C‐terminal regions control the stabilities of BTG1, BTG2, Tob, and Tob2 proteins. In addition, it was found that the respective C‐terminal regions confer instability on green fluorescent protein, a normally stable protein. Thus, it can be concluded that the C‐terminal regions are necessary and sufficient to control the stabilities of BTG1, BTG2, Tob, and Tob2 proteins.
Biglycan isoforms with differences in polysaccharide substitution and core protein in human lung fibroblasts
Authors: Tufvesson Ellen , Malmström Johan , Marko‐Varga György , Westergren‐Thorsson Gunilla
Published: March 2003

Biglycan is widely distributed in the extracellular matrix and is a member of the small proteoglycan family characterized by a core protein with leucine‐rich repeat motifs. We show in this paper for the first time that biglycan from human lung fibroblasts can be expressed as different isoforms. These isoforms can be separated from the predominant form of biglycan by hydrophobic interaction chromatography, where the more hydrophobic isoforms are retarded. The newly found isoforms of biglycan have a smaller core protein substituted with smaller glycosaminoglycan chains, migrating on SDS/PAGE at between 110 and 200 kDa. These molecules were identified as biglycan using MALDI‐TOF MS. Identification of C‐terminal peptides together with glycosylation of the N‐terminal glycosaminoglycan sites excludes the possibility of terminal proteolytic cleavage. The biglycan isoforms are N‐glycosylated, which demonstrates that a lack in N‐glycosylation is not the reason for a smaller core. Two components revealed by RT‐PCR indicate alternative splicing, which could be located in regions of the protein that have not been identified, with the exclusion of sites of glycosylations.

Analyses of glycosaminoglycan chain length of the isoforms show that besides the normally occurring glycosaminoglycan chains, there is a mixture of shorter glycosaminoglycan chains. Structural analysis shows that these glycosaminoglycan chains contain a lower proportion of iduronic acid (61%) relative to glucuronic acid when compared to the glycosaminoglycan chain of the predominant form of biglycan (71%). We can anticipate that variation in structure of biglycan can cause changes in the connective tissue formation depending on its ability to bind matrix molecules, as well as cytokines.
Kinetic studies of human tyrosyl‐DNA phosphodiesterase, an enzyme in the topoisomerase I DNA repair pathway
Authors: Cheng Ting‐Jen , Rey Peter G. , Poon Thomas , Kan Chen‐Chen
Published: March 2003

Tyrosyl‐DNA phosphodiesterase (TDP) cleaves the phosphodiester bond linking the active site tyrosine residue of topoisomerase I with the 3′ terminus of DNA in topoisomerase I–DNA complexes which accumulate during treatment of cancer with camptothecin. In yeast, TDP mutation confers a 1000‐fold hypersensitivity to camptothecin in the presence of an additional mutation of RAD9 gene [Pouliot, J.J., Yao, K.C., Robertson, C.A. & Nash, H.A. (1999) Science
286, 552–555]. Based on the recently solved crystal structure, human TDP belongs to a distinct class within the phospholipase D superfamily in spite of very low sequence homology [Interthal, H., Pouliot, J.J. & Champoux, J.J. (2001) Proc. Natl Acad. Sci. USA
98, 12009–12014, and Davies, D.R., Interthal, H., Champoux, J.J. & Hol, W.G.J. (2002) Structure
10, 237–248]. To understand the enzymatic mechanism of this novel enzyme, and to facilitate inhibitor screening of human TDP, we have expressed and purified recombinant human TDP variants carrying deletions of 1–39 or 1–174 amino acids. Furthermore, a continuous colorimetric assay in a 96‐well format was also developed using p‐nitrophenyl‐thymidine‐3′‐phosphate as substrate. This assay system is able to detect enzymatic activity at enzyme concentrations as low as 15 nm. Purified recombinant human TDPNΔ39 cleaved p‐nitrophenyl‐thymidine‐3′‐phosphate with Km and kcat values of 211.14 ± 23.83 µm and 8.82 ± 0.57 per min in the presence of Mn2+.
Characterization and regulation of yeast Ca2+‐dependent phosphatidylethanolamine‐phospholipase D activity
Authors: Tang Xiaoqing , Waksman Michal , Ely Yona , Liscovitch Mordechai
Published: March 2003

An unconventional phospholipase D (PLD) activity was identified recently in Saccharomyces cerevisiae which is Ca2+‐dependent, preferentially hydrolyses phosphatidylethanolamine (PtdEtn) and phosphatidylserine and does not catalyse a transphosphatidylation with primary short‐chain alcohols. We have characterized the cytosolic and membrane‐bound forms of the yeast PtdEtn‐PLD and examined the regulation of its activity under certain growth, nutritional and stress conditions. Both forms of PtdEtn‐PLD activity were similarly activated by Ca2+ ions in a biphasic manner. Likewise, other divalent cations affected both cytosolic and membrane‐bound forms to the same extent. The yeast PtdEtn‐PLD activity was found to interact with immobilized PtdEtn in a Ca2+‐dependent manner. The partially purified cytosolic form and the salt‐extracted membrane‐bound form of yeast PtdEtn‐PLD exhibited a similar elution pattern on size‐exclusion chromatography, coeluting as low apparent molecular weight peaks. PtdEtn‐PLD activity was stimulated, along with Spo14p/Pld1p activity, upon dilution of stationary phase cultures in glucose, acetate and galactose media, but PtdEtn‐PLD activation was less pronounced. Interestingly, PtdEtn‐PLD activity was found to be elevated by ≈ 40% in sec14ts mutants at the restrictive temperature, whereas in other sec mutants it remained unaffected. The activity of PtdEtn‐PLD was reduced by 30–40% upon addition to the medium of inositol (75 µm) in either wild‐type yeast or spo14Δ mutants and this effect was seen regardless of the presence of choline, suggesting that transcription of the PtdEtn‐PLD gene is down‐regulated by inositol. Finally, exposure of yeast cells to H2O2 resulted in a transient increase in PtdEtn‐PLD activity followed by a profound, nearly 90% decrease in activity. In conclusion, our results indicate that yeast PtdEtn‐PLD activity is highly regulated: the enzyme is acutely activated upon entry into the cell cycle and following inactivation of sec14ts, and is inhibited under oxidative stress conditions. The implications of these findings are discussed.
De‐regulation of d‐3‐phosphoglycerate dehydrogenase by domain removal
Authors: Bell Jessica K. , Pease Paul J. , Bell J. Ellis , Grant Gregory A. , Banaszak Leonard J.
Published: March 2003

Escherichia coli 3‐phosphoglycerate dehydrogenase (PGDH) catalyzes the first step in serine biosynthesis, and is allosterically inhibited by serine. Structural studies revealed a homotetramer in which the quaternary arrangement of subunits formed an elongated ellipsoid. Each subunit consisted of three domains: nucleotide, substrate and regulatory. In PGDH, extensive interactions are formed between nucleotide binding domains. A second subunit–subunit interaction occurs between regulatory domains creating an extended β sheet. The serine‐binding sites overlap this interface. In these studies, the nucleotide and substrate domains (NSDs) were subcloned to identify changes in both catalytic and physical properties upon removal of a subunit–subunit interface. The NSDs did not vary significantly from PGDH with respect to kinetic parameters with the exception that serine no longer had an effect on catalysis. Temperature dependent dynamic light scattering (DLS) revealed the NSDs aggregated > 5 °C before PGDH, indicating decreased stability. DLS and gel filtration studies showed that the truncated enzyme formed a tetramer. This result negated the hypothesis that the removal of the regulatory domain would create an enzyme mimic of the unregulated, closely related dimeric enzymes. Expression of the regulatory domain, to study conformational changes induced by serine binding, yielded a product that by CD spectra contained stable secondary structure. DLS and pulsed field gradient NMR studies of the regulatory domain showed the presence of higher oligomers instead of the predicted dimer. We have concluded that the removal of the regulatory domain is sufficient to eliminate serine inhibition but does not have the expected effect on the quaternary structure.
Structures and mode of membrane interaction of a short α helical lytic peptide and its diastereomer determined by NMR, FTIR, and fluorescence spectroscopy
Authors: Oren Ziv , Ramesh Jagannathan , Avrahami Dorit , Suryaprakash N. , Shai Yechiel , Jelinek Raz
Published: March 2003

The interaction of many lytic cationic antimicrobial peptides with their target cells involves electrostatic interactions, hydrophobic effects, and the formation of amphipathic secondary structures, such as α helices or β sheets. We have shown in previous studies that incorporating ≈ 30%d‐amino acids into a short α helical lytic peptide composed of leucine and lysine preserved the antimicrobial activity of the parent peptide, while the hemolytic activity was abolished. However, the mechanisms underlying the unique structural features induced by incorporating d‐amino acids that enable short diastereomeric antimicrobial peptides to preserve membrane binding and lytic capabilities remain unknown. Inthis study, we analyze in detail the structuresofa modelamphipathic α helical cytolytic peptide KLLLKWLL KLLK‐NH2 and its diastereomeric analog and their interactions with zwitterionic and negatively charged membranes. Calculations based on high‐resolution NMR experiments in dodecylphosphocholine (DPCho) and sodium dodecyl sulfate (SDS) micelles yield three‐dimensional structures of both peptides. Structural analysis reveals that the peptides have an amphipathic organization within both membranes. Specifically, the α helical structure of theL‐type peptide causes orientation of the hydrophobic andpolar amino acids onto separate surfaces, allowing interactions with both the hydrophobic core of the membrane andthe polar head group region. Significantly, despite the absence of helical structures, the diastereomer peptide analog exhibits similar segregation between the polar and hydrophobic surfaces. Further insight into the membrane‐binding properties of the peptides and their depth of penetration into the lipid bilayer has been obtained through tryptophan quenching experiments using brominated phospholipids and the recently developed lipid/polydiacetylene (PDA) colorimetric assay. The combined NMR, FTIR, fluorescence, and colorimetric studies shed light on the importance of segregation between the positive charges and the hydrophobic moieties on opposite surfaces within the peptides for facilitating membrane binding and disruption, compared to the formation of α helical or β sheet structures.
Directed evolution of a glutaryl acylase into an adipyl acylase
Authors: Sio Charles F. , Riemens Anja M. , van der Laan Jan‐Metske , Verhaert Raymond M.D. , Quax Wim J.
Published: March 2003

Semi‐synthetic cephalosporin antibiotics belong to the top 10 of most sold drugs, and are produced from 7‐aminodesacetoxycephalosporanic acid (7‐ADCA). Recently new routes have been developed which allow for the production of adipyl‐7‐ADCA by a novel fermentation process. To complete the biosynthesis of 7‐ADCA a highly active adipyl acylase is needed for deacylation of the adipyl derivative. Such an adipyl acylase can be generated from known glutaryl acylases.

The glutaryl acylase of Pseudomonas SY‐77 was mutated in a first round by exploration mutagenesis. For selection the mutants were grown on an adipyl substrate. The residues that are important to the adipyl acylase activity were identified, and in a second round saturation mutagenesis of this selected stretch of residues yielded variants with a threefold increased catalytic efficiency. The effect of the mutations could be rationalized on hindsight by the 3D structure of the acylase.

In conclusion, the substrate specificity of a dicarboxylic acid acylase was shifted towards adipyl‐7‐ADCA by a two‐step directed evolution strategy. Although derivatives of the substrate were used for selection, mutants retained activity on the β‐lactam substrate. The strategy herein described may be generally applicable to all β‐lactam acylases.
Association of the thyrotropin receptor with calnexin, calreticulin and BiP
Authors: Siffroi‐Fernandez Sandrine , Giraud Annie , Lanet Jeanne , Franc Jean‐Louis
Published: March 2003

The thyrotropin receptor (TSHR) is a member of the G protein‐coupled receptor superfamily. It has by now been clearly established that the maturation of the glycoproteins synthesized in the endoplasmic reticulum involves interactions with molecular chaperones, which promote the folding and assembly of the glycoproteins. In this study, we investigated whether calnexin (CNX), calreticulin (CRT) and BiP, three of the main molecular chaperones present in the endoplasmic reticulum, interact with the TSHR and what effects these interactions might have on the folding of the receptor. In the first set of experiments, we observed that in a K562 cell line expressing TSHR, about 50% of the receptor synthesized was degraded by the proteasome after ubiquitination. In order to determine whether TSHR interact with CNX, CRT and BiP, coimmunoprecipitation experiments were performed. TSHR was found to be associated with all three molecular chaperones. To study the role of the interactions between CNX and CRT and the TSHR, we used castanospermine, a glucosidase I and II inhibitor that blocks the interactions between these chaperones and glycoproteins. In K562 cells expressing the TSHR, these drugs led to a faster degradation of the receptor, which indicates that these interactions contribute to stabilizing the receptor after its synthesis. The overexpression of calnexin and calreticulin in these cells stabilizes the receptor during the first hour after its synthesis, whereas the degradation of TSHR increased in a cell line overexpressing BiP and the quantity of TSHR able to acquire complex type oligosaccharides decreased. These results show that calnexin, calreticulin and BiP all interact with TSHR and that the choice made between these two chaperone systems is crucial because each of them has distinct effects on the folding and stability of this receptor at the endoplasmic reticulum level.