bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of cleavage and shedding in human thyrotropin receptor function and trafficking
Authors: Quellari Mylène , Desroches Agnès , Beau Isabelle , Beaudeux Emmanuelle , Misrahi Micheline
Published: August 2003

The thyrotropin receptor (TSHR) undergoes a cleavage at the cell membrane, leading to a heterodimer, comprising an α extracellular and a β‐transmembrane and intracellular subunits, held together by disulfide bonds. Moreover, part of the α‐subunit of the receptor is shed from thyroid and transfected L cells. To understand the role of cleavage and shedding, we constructed deletion mutants starting, respectively, at the most N‐terminal (S314), and C‐terminal (L378) cleavage sites previously mapped, corresponding to free β1 or β2‐subunits without further modification of receptor structure. Functional studies performed in COS‐7 cells showed that both mutants display an increased basal activation of the cAMP pathway when compared with the wild‐type receptor. By contrast, deletion of almost the entire extracellular domain of the receptor (TM409 mutant) totally impairs receptor function, thus confirming a role of the juxtamembrane extracellular region in receptor function. The β1 mutant receptor exhibited an increased internalization when compared with the hormone‐activated holoreceptor. Furthermore, no recycling was observed in the case of the β1 mutant receptor. These observations strongly argue for a different conformation between the receptor activated by cleavage and shedding on the one hand, and the receptor activated by the ligand on the other hand. Cleavage and shedding of a receptor already activated by a transmembrane activating mutation M453T further increase its activity, showing that the extracellular domain still exerts a negative effect in the M453T holoreceptor. An increased internalization of the M453T receptor was observed when compared with the wild‐type receptor, which was increased further in the corresponding truncated β1‐M453T receptor. Thus cleavage and shedding yield TSHR activation but also increase internalization of the free β‐subunits of the receptor, the latter mechanism limiting simultaneously excessive receptor signaling. The combined effects may be responsible for the limited basal constitutive activation of the cAMP pathway that is detected for the TSHR.
Uptake and metabolism of [3H]anandamide by rabbit platelets
Authors: Fasia Lambrini , Karava Vivi , Siafaka‐Kapadai Athanassia
Published: August 2003

Anandamide is an endogenous ligand for cannabinoid receptor and its protein‐mediated transport across cellular membranes has been demonstrated in cells derived from brain as well as in cells of the immune system. This lipid is inactivated via intracellular degradation by a fatty acid amidohydrolase (FAAH). In the present study, we report that rabbit platelets, in contrast to human platelets, do not possess a carrier‐mediated mechanism for the transport of [3H]anandamide into the cell, i.e. cellular uptake was not temperature dependent and its accumulation was not saturable. This endocannabinoid appears to enter the cell by simple diffusion. Once taken up by rabbit platelets, [3H]anandamide was rapidly metabolized into compounds which were secreted into the medium. Small amounts of free arachidonic acid as well as phospholipids were amongst the metabolic products. FAAH inhibitors did not decrease anandamide uptake, whereas these compounds inhibited anandamide metabolism. In conclusion, anandamide is rapidly taken up by rabbit platelets and metabolized mainly into water‐soluble metabolites. Interestingly, the present study also suggests the absence of a transporter for anandamide in these cells.
Anti‐HIV‐1 activity of 3‐deaza‐adenosine analogs
Authors: Gordon Richard K. , Ginalski Krzysztof , Rudnicki Witold R. , Rychlewski Leszek , Pankaskie Marvin C. , Bujnicki Janusz M. , Chiang Peter K.
Published: August 2003

Eight adenosine analogs, 3‐deaza‐adenosine (DZA), 3‐deaza‐(±)aristeromycin (DZAri), 2′,3′‐dideoxy‐adenosine (ddAdo), 2′,3′‐dideoxy‐3‐deaza‐adenosine (ddDZA), 2′,3′‐dideoxy‐3‐deaza‐(±)aristeromycin (ddDZAri), 3‐deaza‐5′‐(±)noraristeromycin (DZNAri), 3‐deaza‐neplanocin A (DZNep), and neplanocin A (NepA), were tested as inhibitors of human placenta S‐adenosylhomocysteine (AdoHcy) hydrolase. The order of potency for the inhibition of human placental AdoHcy hydrolase was: DZNep ≈ NepA >> DZAri ≈ DZNAri > DZA >> ddAdo ≈ ddDZA ≈ ddDZAri. These same analogs were examined for their anti‐HIV‐1 activities measured by the reduction in p24 antigen produced by 3′‐azido‐3′‐deoxythymidine (AZT)‐sensitive HIV‐1 isolates, A012 and A018, in phytohemagglutinin‐stimulated peripheral blood mononuclear (PBMCs) cells. Interestingly, DZNAri and the 2′,3′‐dideoxy 3‐deaza‐nucleosides (ddAdo, ddDZAri, and ddDZA) were only marginal inhibitors of p24 antigen production in HIV‐1 infected PBMC. DZNAri is unique because it is the only DZA analog with a deleted methylene group that precludes anabolic phosphorylation. In contrast, the other analogs were potent inhibitors of p24 antigen production by both HIV‐1 isolates. Thus it was postulated that these nucleoside analogs could exert their antiviral effect via a combination of anabolically generated nucleotides (with the exception of DZNAri), which could inhibit reverse transcriptase or other viral enzymes, and the inhibition of viral or cellular methylation reactions. Additionally, QSAR‐like models based on the molecular mechanics (MM) were developed to predict the order of potency of eight adenosine analogs for the inhibition of human AdoHcy hydrolase. In view of the potent antiviral activities of the DZA analogs, this approach provides a promising tool for designing and screening of more potent AdoHcy hydrolase inhibitors and antiviral agents.
Interconversions among four M‐intermediates in the bacteriorhodopsin photocycle
Authors: Hendler Richard W. , Bose Salil
Published: August 2003

Halobacterium salinarum displays four distinct kinetic forms of M‐intermediate in its bacteriorhodopsin photocycle. In wild‐type, there are mainly two species with time constants near 2 and 5 ms. Under various kinds of stress, two other species arise with time constants near 10 and 70 ms. We show that these four species are interconvertible. Increases in membrane hydrophobicity convert the slower to faster forms. Perturbations caused by Triton X‐100 or mutations convert faster to slower forms. The fastest form requires a hydrophobic membrane environment near a ring of four charged aspartate residues in the trimer, namely Asp36, Asp38, Asp102, and Asp104 in the cytoplasmic loop regions. Interconversions of the 2‐ms and 5‐ms species of the wild‐type are accomplished by pH‐changes. The potential significance of these findings is discussed.
Systematic quantification of complex metabolic flux networks using stable isotopes and mass spectrometry
Authors: Klapa Maria I. , Aon Juan‐Carlos , Stephanopoulos Gregory
Published: August 2003

Metabolic fluxes provide a detailed metric of the cellular metabolic phenotype. Fluxes are estimated indirectly from available measurements and various methods have been developed for this purpose. Of particular interest are methods making use of stable isotopic tracers as they enable the estimation of fluxes at a high resolution. In this paper, we present data validating the use of mass spectrometry (MS) for the quantification of complex metabolic flux networks. In the context of the lysine biosynthesis flux network of Corynebacterium glutamicum (ATCC 21799) under glucose limitation in continuous culture, operating at 0.1·h−1 after the introduction of 50% [1‐13C]glucose, we deploy a bioreaction network analysis methodology for flux determination from mass isotopomer measurements of biomass hydrolysates, while thoroughly addressing the issues of measurement accuracy, flux observability and data reconciliation. The analysis enabled the resolution of the involved anaplerotic activity of the microorganism using only one labeled substrate, the determination of the range of most of the exchange fluxes and the validation of the flux estimates through satisfaction of redundancies. Specifically, we determined that phosphoenolpyruvate carboxykinase and synthase do not carry flux at these experimental conditions and identified a high futile cycle between oxaloacetate and pyruvate, indicating a highly active in vivo oxaloacetate decarboxylase. Both results validated previous in vitro activity measurements. The flux estimates obtained passed the χ2 statistical test. This is a very important result considering that prior flux analyses of extensive metabolic networks from isotopic measurements have failed criteria of statistical consistency.
Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70
Authors: Nuttall Stewart D. , Krishnan Usha V. , Doughty Larissa , Pearson Kylie , Ryan Michael T. , Hoogenraad Nicholas J. , Hattarki Meghan , Carmichael Jennifer A. , Irving Robert A. , Hudson Peter J.
Published: August 2003

The new antigen receptor (IgNAR) from sharks is a disulphide bonded dimer of two protein chains, each containing one variable and five constant domains, and functions as an antibody. In order to assess the antigen‐binding capabilities of isolated IgNAR variable domains (VNAR), we have constructed an in vitro library incorporating synthetic CDR3 regions of 15–18 residues in length. Screening of this library against the 60 kDa cytosolic domain of the 70 kDa outer membrane translocase receptor from human mitochondria (Tom70) resulted in one dominant antigen‐specific clone (VNAR 12F‐11) after four rounds of in vitro selection. VNAR 12F‐11 was expressed into the Escherichia coli periplasm and purified by anti‐FLAG affinity chromatography at yields of 3 mg·L−1. Purified protein eluted from gel filtration columns as a single monomeric protein and CD spectrum analysis indicated correct folding into the expected β‐sheet conformation. Specific binding to Tom70 was demonstrated by ELISA and BIAcore (Kd = 2.2 ± 0.31 × 10−9 m−1) indicating that these VNAR domains can be efficiently displayed as bacteriophage libraries, and selected against target antigens with an affinity and stability equivalent to that obtained for other single domain antibodies. As an initial step in producing ‘intrabody’ variants of 12F‐11, the impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed. High affinity binding was only retained in the wild‐type protein, which combined with our inability to affinity mature 12F‐11, suggests that this particular VNAR is critically dependent upon precise CDR loop conformations for its binding affinity.
Relationships between the ethanol utilization (alc) pathway and unrelated catabolic pathways in Aspergillus nidulans
Authors: Flipphi Michel , Kocialkowska Janina , Felenbok Béatrice
Published: August 2003

The ethanol utilization pathway in Aspergillus nidulans is a model system, which has been thoroughly elucidated at the biochemical, genetic and molecular levels. Three main elements are involved: (a) high level expression of the positively autoregulated activator AlcR; (b) the strong promoters of the structural genes for alcohol dehydrogenase (alcA) and aldehyde dehydrogenase (aldA); and (c) powerful activation of AlcR by the physiological inducer, acetaldehyde, produced from growth substrates such as ethanol and l‐threonine. We have previously characterized the chemical features of direct inducers of the alc regulon. These studies allowed us to predict which type of carbonyl compounds might induce the system. In this study we have determined that catabolism of different amino acids, such as l‐valine, l‐isoleucine, l‐arginine and l‐proline, produces aldehydes that are either not accumulated or fail to induce the alc system. On the other hand, catabolism of d‐galacturonic acid and putrescine, during which aldehydes are transiently accumulated, gives rise to induction of the alc genes. We show that the formation of a direct inducer from carboxylic esters does not depend on alcA‐encoded alcohol dehydrogenase I or on AlcR, and suggest that a cytochrome P450 might be responsible for the initial formation of a physiological aldehyde inducer.
Re‐evaluation of intramolecular long‐range electron transfer between tyrosine and tryptophan in lysozymes
Authors: Stuart‐Audette Marilyne , Blouquit Yves , Faraggi Moshe , Sicard‐Roselli Cécile , Houée‐Levin Chantal , Jollès Pierre
Published: August 2003

One‐electron oxidation of six different c‐type lysozymes from hen egg white, turkey egg white, human milk, horse milk, camel stomach and tortoise was studied by gamma‐ and pulse‐radiolysis. In the first step, one tryptophan side chain is oxidized to indolyl free radical, which is produced quantitatively. As shown already, the indolyl radical subsequently oxidizes a tyrosine side chain to the phenoxy radical in an intramolecular reaction. However this reaction is not total and its stoichiometry depends on the protein. Rate constants also vary between proteins, from 120·s−1 to 1000·s−1 at pH 7.0 and room temperature [extremes are hen and turkey egg white (120·s−1) and human milk (1000·s−1)]. In hen and turkey egg white lysozymes we show that another reactive site is the Asn103–Gly104 peptidic bond, which gets broken radiolytically. Tryptic digestion followed by HPLC separation and identification of the peptides was performed for nonirradiated and irradiated hen lysozyme. Fluorescence spectra of the peptides indicate that Trp108 and/or 111 remain oxidized and that Tyr20 and 53 give bityrosine. Tyr23 appears not to be involved in the process. Thus new features of long‐range intramolecular electron transfer in proteins appear: it is only partial and other groups are involved which are silent in pulse radiolysis.
Synthesis and characterization of Pi4, a scorpion toxin from Pandinus imperator that acts on K+ channels
Authors: M'Barek Sarrah , Mosbah Amor , Sandoz Guillaume , Fajloun Ziad , Olamendi‐Portugal Timoteo , Rochat Hervé , Sampieri François , Guijarro J. Iñaki , Mansuelle Pascal , Delepierre Muriel , De Waard Michel , Sabatier Jean‐Marc
Published: August 2003

Pi4 is a 38‐residue toxin cross‐linked by four disulfide bridges that has been isolated from the venom of the Chactidae scorpion Pandinus imperator. Together with maurotoxin, Pi1, Pi7 and HsTx1, Pi4 belongs to the α KTX6 subfamily of short four‐disulfide‐bridged scorpion toxins acting on K+ channels. Due to its very low abundance in venom, Pi4 was chemically synthesized in order to better characterize its pharmacology and structural properties. An enzyme‐based cleavage of synthetic Pi4 (sPi4) indicated half‐cystine pairings between Cys6–Cys27, Cys12–32, Cys16–34 and Cys22–37, which denotes a conventional pattern of scorpion toxin reticulation (Pi1/HsTx1 type). In vivo, sPi4 was lethal after intracerebroventricular injection to mice (LD50 of 0.2 µg per mouse). In vitro, addition of sPi4 onto Xenopus laevis oocytes heterologously expressing various voltage‐gated K+ channel subtypes showed potent inhibition of currents from rat Kv1.2 (IC50 of 8 pm) and Shaker B (IC50 of 3 nm) channels, whereas no effect was observed on rat Kv1.1 and Kv1.3 channels. The sPi4 was also found to compete with 125I‐labeled apamin for binding to small‐conductance Ca2+‐activated K+ (SK) channels from rat brain synaptosomes (IC50 value of 0.5 µm). sPi4 is a high affinity blocker of the Kv1.2 channel. The toxin was docked (Bigger program) on the Kv channel using the solution structure of sPi4 and a molecular model of the Kv1.2 channel pore region. The model suggests a key role for residues Arg10, Arg19, Lys26 (dyad), Ile28, Lys30, Lys33 and Tyr35 (dyad) in the interaction and the associated blockage of the Kv1.2 channel.
A thermoacidophilic endoglucanase (CelB) from Alicyclobacillus acidocaldarius displays high sequence similarity to arabinofuranosidases belonging to family 51 of glycoside hydrolases
Authors: Eckert Kelvin , Schneider Erwin
Published: August 2003

A 100‐kDa protein with endoglucanase activity was purified from Triton X‐100 extract of cells of the thermoacidophilic Gram‐positive bacterium Alicyclobacillus acidocaldarius. The enzyme exhibited activity towards carboxy methyl cellulose and oat spelt xylan with pH and temperature optima of 4 and 80 °C, respectively. Cloning and nucleotide sequence analysis of the corresponding gene (celB) revealed an ORF encoding a preprotein of 959 amino acids which is consistent with an extracellular localization. Purified recombinant CelB and a variant lacking the C‐terminal 203 amino acid residues (CelBtrunc) displayed similar enzymatic properties as the wild‐type protein. Analysis of product formation suggested an endo mode of action. Remarkable stability was observed at pH values between 1 and 7 and 60% of activity were retained after incubation for 1 h at 80 °C. CelB displayed homology to members of glycoside hydrolase family 51, being only the second entry with activity typical of an endoglucanase but lacking activity on p‐nitrophenyl‐α‐l‐arabinofuranoside (pNPAraf). Highest sequence similarity was found towards the other endoglucanase F from Fibrobacter succinogenes (EGF), forming a distinct group in the phylogenetic tree of this family. Analysis of the amino acid composition of the catalytic domains demonstrated that CelB contains fewer charged amino acids than its neutrophilic counterparts, which is in line with adaptation to low pH. Wild‐type and full‐length recombinant CelB were soluble only in Triton X‐100. In contrast, CelBtrunc was completely water soluble, suggesting a role of the C‐terminal region in cell association. This C‐terminal hydrophobic region displayed local sequence similarities to an α‐amylase from the same organism.
Does different orientation of the methoxy groups of ubiquinone‐10 in the reaction centre of Rhodobacter sphaeroides cause different binding at QA and QB?
Authors: Remy André , Boers Rutger B. , Egorova‐Zachernyuk Tatiana , Gast Peter , Lugtenburg Johan , Gerwert Klaus
Published: August 2003

The different roles of ubiquinone‐10 (UQ10) at the primary and secondary quinone (QA and QB) binding sites of Rhodobacter sphaeroides R26 reaction centres are governed by the protein microenvironment. The 4C=O carbonyl group of QA is unusually strongly hydrogen‐bonded, in contrast to QB. This asymmetric binding seems to determine their different functions. The asymmetric hydrogen‐bonding at QA can be caused intrinsically by distortion of the methoxy groups or extrinsically by binding to specific amino‐acid side groups. Different X‐ray‐based structural models show contradictory orientations of the methoxy groups and do not provide a clear picture. To elucidate if distortion of the methoxy groups induces this hydrogen‐bonding, their (ring‐)C‐O vibrations were assigned by use of site‐specifically labelled [5‐13C]UQ10 and [6‐13C]UQ10 reconstituted at either the QA or the QB binding site. Two infrared bands at 1288 cm−1 and 1264 cm−1 were assigned to the methoxy vibrations. They did not shift in frequency at either the QA or QB binding sites, as compared with unbound UQ10. As the frequencies of these vibrations and their coupling are sensitive to the conformations of the methoxy groups, different conformations of the C(5) and C(6) methoxy groups at the QA and QB binding sites can now be excluded. Both methoxy groups are oriented out of plane at QA and QB. Therefore, hydrogen‐bonding to His M219 combined with electrostatic interactions with the Fe2+ ion seems to determine the strong asymmetric binding of QA.
Fluorescent and colored trinitrophenylated analogs of ATP and GTP
Authors: Hiratsuka Toshiaki
Published: August 2003

Fluorescent and colored trinitrophenylated (TNP) analogs of ATP and GTP can interact with nucleotide‐requiring enzymes and proteins as a substitute for the parent nucleotide. These analogs have strong binding affinities for most nucleotide‐requiring systems. Their bindings are easily detected by absorption and fluorescence changes in the visible region. Recent years have seen dramatic developments in the application of the TNP nucleotide analogs as spectroscopic probes for the study on the nucleotide‐interacting properties of various enzymes and proteins including their mutants. This review is intended as a broad overview of currently extensively used applications of the nucleotide analogs in various biological systems.
The CRIPTO/FRL‐1/CRYPTIC (CFC) domain of human Cripto
Authors: Foley Susan F. , van Vlijmen Herman W. T. , Boynton Raymond E. , Adkins Heather B. , Cheung Anne E. , Singh Juswinder , Sanicola Michele , Young Carmen N. , Wen Dingyi
Published: August 2003

The disulfide structure of the CRIPTO/FRL‐1/CRYPTIC (CFC) domain of human Cripto protein was determined by a combination of enzymatic and chemical fragmentation, followed by chromatographic separation of the fragments, and characterization by mass spectrometry and N‐terminal sequencing. These studies showed that Cys115 forms a disulfide bond with Cys133, Cys128 with Cys149, and Cys131 with Cys140. Protein database searching and molecular modeling revealed that the pattern of disulfide linkages in the CFC domain of Cripto is the same as that in PARS intercerebralis major Peptide C (PMP‐C), a serine protease inhibitor, and that the EGF‐CFC domains of Cripto are predicted to be structurally homologous to the EGF‐VWFC domains of the C‐terminal extracellular portions of Jagged 1 and Jagged 2. Biochemical studies of the interactions of ALK4 with the CFC domain of Cripto by fluorescence‐activated cell sorter analysis indicate that the CFC domain binds to ALK4 independent of the EGF domain. A molecular model of the CFC domain of Cripto was constructed based on the nuclear magnetic resonance structure of PMP‐C. This model reveals a hydrophobic patch in the domain opposite to the presumed ALK4 binding site. This hydrophobic patch may be functionally important for the formation of intra or intermolecular complexes.
Prevalent conformations and subunit exchange in the biologically active apoptin protein multimer
Authors: Leliveld Sirik R. , Noteborn Mathieu H. M. , Abrahams Jan Pieter
Published: August 2003

Recombinant, bacterially expressed apoptin protein induces apoptosis in human tumour cell lines but not in normal cells, mimicking the behaviour of ectopically expressed apoptin. Recombinant apoptin is isolated exclusively as a highly stable multimeric complex of 30–40 monomers, with little, if any, α‐helical and β‐sheet structure. Despite its apparent disorder, multimeric apoptin is biologically active. Here, we present evidence that most of the apoptin moieties within the complex may well share a similar conformation. Furthermore, the multimer has extensive and uniform hydrophobic patches and conformationally stable domains. Only a small fraction of apoptin subunits can exchange between multimers under physiologically relevant conditions. These results prompt a model in which the apoptin multimer has a highly stable core of nonexchangeable subunits to which exchangeable subunits are attached through hydrophobic interactions. In combination with previous findings, our results lead us to propose that the stable core of apoptin is the biologically relevant structure.
Effect of mutations at Glu160 and Val198 on the thermostability of lactate oxidase
Authors: Minagawa Hirotaka , Shimada Jiro , Kaneko Hiroki
Published: August 2003

We have obtained two types of thermostable mutant lactate oxidase – one that exhibited an E‐to‐G point mutation at position 160 (E160G) through error‐prone PCR‐based random mutagenesis, and another that exhibited an E‐to‐G mutation at position 160 and a V‐to‐I mutation at position 198 (E160G/V198I) through DNA shuffling‐based random mutagenesis – both of which we have previously reported. Our molecular modeling of lactate oxidase suggests that the substitution of G for E at position 160 reduces the electrostatic repulsion between the negative charges of E160 and E130 in the (β/α)8 barrel structure, but a thermal‐inactivation experiment on the five kinds of single‐mutant lactate oxidase at position 160 (E160A, E160Q, E160H, E160R, and E160K) showed that the side‐chain volume of the amino acid at position 160 mainly contributes to the thermostability of lactate oxidase. We also produced V198I single‐mutant lactate oxidase through site‐directed mutagenesis, and analysed the thermostability of wild‐type, V198I, E160G, and E160G/V198I lactate oxidase enzymes. The half‐life of E160G/V198I lactate oxidase at 70 °C was about three times longer than that of E160G lactate oxidase, and was about 20 times longer than that of wild‐type lactate oxidase. In contrast, the thermostability of the V198I lactate oxidase was almost identical to that of wild‐type lactate oxidase. This indicates that the V198I mutation alone does not affect lactate oxidase thermostability, but does affect it when combined with the E160G mutation.
First evidence of catalytic mediation by phenolic compounds in the laccase‐induced oxidation of lignin models
Authors: d'Acunzo Francesca , Galli Carlo
Published: August 2003

The sulfonephthalein indicator, phenol red, exhibits an unusually slow rate of oxidation by laccase from Poliporus pinsitus, in spite of the fact that it is a phenol and therefore a natural substrate for this phenoloxidase enzyme. Nevertheless, after prolonged exposure to laccase (24 h) phenol red is oxidized by more than 90%. We found that phenol red, which can be oxidatively converted into a resonance‐stabilized phenoxy radical, performs as a mediator in the laccase‐catalyzed oxidation of a nonphenolic substrate (4‐methoxybenzyl alcohol) and also of a hindered phenol (2,4,6‐tri‐tert‐butylphenol). In particular, phenol red was found to be at least 10 times more efficient than 3‐hydroxyanthranilate (a reported natural phenolic mediator of laccase) in the oxidation of 4‐methoxybenzyl alcohol. Other phenols, which do not bear structural analogies to phenol red, underwent rapid degradation and did not perform as laccase mediators. On the other hand, several variously substituted sulfonephthaleins, of different pK2 values, mediated the laccase catalysis, the most efficient being dichlorophenol red, which has the lowest pK2 of the series. The mediating efficiency of phenol red and dichlorophenol red was found to be pH dependent, as was their oxidation Ep value (determined by cyclic voltammetry). We argue that the relative abundance of the phenoxy anion, which is easier to oxidize than the protonated phenol, may be one of the factors determining the efficiency of a phenolic mediator, together with its ability to form relatively stable oxidized intermediates that react with the desired substrate before being depleted in undesired routes.
Analysis of the effect of potato carboxypeptidase inhibitor pro‐sequence on the folding of the mature protein
Authors: Bronsoms Sílvia , Villanueva Josep , Canals Francesc , Querol Enrique , Aviles Francesc X.
Published: August 2003

Protein folding can be modulated in vivo by many factors. While chaperones act as folding catalysts and show broad substrate specificity, some pro‐peptides specifically facilitate the folding of the mature protein to which they are bound. Potato carboxypeptidase inhibitor (PCI), a 39‐residue protein carboxypeptidase inhibitor, is synthesized in vivo as a precursor protein that includes a 27‐residue N‐terminal and a seven‐residue C‐terminal pro‐regions. In this work the disulfide‐coupled folding of mature PCI in vitro has been compared with that of the same protein extended with either the N‐terminal pro‐sequence (ProNtPCI) or both N‐ and C‐terminal pro‐sequences (ProPCI), and also with the N‐terminal pro‐sequence in trans (ProNt + PCI). No significant differences can be observed in the folding kinetics or efficiencies of all these molecules. In addition, in vivo folding studies in Escherichia coli have been performed using wild‐type PCI and three PCI mutant forms with and without the N‐terminal pro‐sequence, the mutations had been previously reported to affect folding of the PCI mature form. The extent to which the ‘native‐like’ form was secreted to the media by each construction was not affected by the presence of the N‐terminal pro‐sequence. These results indicate that PCI does not depend on the N‐terminal pro‐sequence for its folding in both, in vitro and in vivo in E. coli. However, structural analysis by spectroscopy, hydrogen exchange and limited proteolysis by mass spectrometry, indicate the capability of such N‐terminal pro‐sequence to fold within the precursor form.
The phosphorylation pattern of human αs1‐casein is markedly different from the ruminant species
Authors: Sørensen Esben S. , Møller Lise , Vinther Maria , Petersen Torben E. , Rasmussen Lone K.
Published: August 2003

Caseins are highly phosphorylated milk proteins assembled in large colloidal structures termed micelles. In the milk of ruminants, αs1‐casein has been shown to be extensively phosphorylated. In this report we have determined the phosphorylation pattern of human αs1‐casein by a combination of matrix‐assisted laser desorption mass spectrometry and amino acid sequence analysis. Three phosphorylation variants were identified. A nonphosphorylated form, a variant phosphorylated at Ser18 and a variant phosphorylated at Ser18 and Ser26. Both phosphorylation sites are located in the amino acid recognition sequence of the mammary gland casein kinase. Notably, no phosphorylations were observed in the conserved region covering residues Ser70–Glu78, which is extensively phosphorylated in the ruminant αs1‐caseins.
Frequencies and spatial patterns of white hypovirulent and pigmented strains of Cryphonectria parasitica within blight‐controlled cankers on grafted American chestnut trees 15–16 years after inoculation
Authors: Griffin G. J.
Published: August 2003

The frequencies and spatial patterns of white and pigmented strains of Cryphonectria parasitica were investigated within cankers in a zone on grafted American chestnut trees inoculated with white (European) and pigmented hypovirulent strains (H‐inoculated zone) 15–16 years earlier. Six 7 × 7 lattice plots (each 17.8 × 17.8 cm) were established on cankers in the H‐inoculated zone of the grafts. Assays of 49 bark cores per lattice indicated that 35.3% of 306 C. parasitica isolates recovered from the six lattice plots were white. The white isolates had a random pattern, potentially favorable to biocontrol, within the highly superficial cankers, based on join‐count statistics of the six lattice plots. Pigmented isolates dominated the C. parasitica population, and virulence trials on American chestnut sprouts indicated 36% of the pigmented isolates from the H‐inoculated zone were hypovirulent and 27% were virulent. Most (84.3%) pigmented isolates in a bark core could not be converted to the white phenotype in vitro by white isolates in the same bark core. Five of six lattice plots had a random pattern of pigmented isolates, based on join‐count statistics. The sixth lattice plot was composed of an aggregate of 36 lattice cells (area = 232 cm2) containing 12 pigmented vegetative compatability (vc) groups of C. parasitica, which were interwoven in the lattice as a mosaic of thread‐like forms, blocks, or ‘islands’ 32 cm2 or less in area for each vc group. Hypotheses are advanced to explain why virulent pigmented strains persist in blight‐controlled cankers of the H‐inoculated zone but do not kill the vascular cambium.
Apoplastic and total peroxidase activities in Scots pine needles at subarctic polluted sites
Authors: Roitto M. , Ahonen‐Jonnarth U. , Lamppu J. , Huttunen S.
Published: August 2003

A gradient survey was carried out in order to compare peroxidase activity in Scots pine (Pinus sylvestris) needles in relation to distance from the industrial centre of Monchegorsk, on the Kola Peninsula in north‐western Russia. Apoplastic and total peroxidase activity and sulphur (S), nickel (Ni) and copper (Cu) content in the needles of mature trees were measured on seven plots located between 10 and 110 km from the pollution source. Peroxidase activities in both current‐ and previous‐year needles increased towards the smelters and showed a positive correlation with needle S, Cu and Ni concentrations. Total peroxidase activities showed a more obvious relationship to the pollution gradient in winter than in autumn. The element contents in the current year needles averaged 1649 ppm (S), 128 ppm (Ni) and 118 ppm (Cu) close to the smelters, 1212 ppm (S), 37 ppm (Ni) and 67 ppm (Cu) at a distance of 40 km and 831 ppm (S), 7 ppm (Ni) and 1 ppm (Cu) at the most distant sampling plot.This study showed that both the apoplastic and total peroxidase activities responded to heavy metal and sulphur pollution up to 40 km from the smelters in winter, which indicated an increased oxidative stress in this area. The harsh climate conditions and the high pollution levels may have had additive effects. However, as peroxidases are considered a general indicator of stress, it is not possible to evaluate the extent to which single pollutants contribute to this enzyme activity.
Varietal mixture of poplar clones: Effects on infection by Melampsora larici‐populina and on plant growth
Authors: Miot By S. , Frey P. , Pinon J.
Published: August 2003

The effects of poplar varietal mixture on infection levels caused by Melampsora larici‐populina and on plant growth were studied during two consecutive growing seasons. The varietal mixture consisted of an intimate mixture of three poplar clones, Populus x interamericana‘Boelare’, ‘Rap’ and Populus candicans‘NNT’, expressing different race‐specific resistances. Infection levels of M. larici‐populina were assessed in monoclonal and polyclonal plots. Plant heights were determined at the end of each growing season. Infection levels in the growing season of the first year were not significantly different between monoclonal and polyclonal plots at the end of the epidemic. During the second growing season, a naturally occurring complex pathotype, able to infect clones ‘Boelare’ and ‘Rap’, invaded some plots prematurely and changed the composition of pathogen population. During this season, four infection assessments were conducted. Infection reduction in polyclonal plots compared with monoclonal plots was observed on ‘Boelare’ and P. candicans plants. Nevertheless, no significant difference was observed for growth between monoclonal and polyclonal plots. Biological factors which may be involved in the reduction of infection levels in polyclonal plots are dilution of susceptible individuals, physical barriers caused by resistant plants and cross‐protection. A high auto‐infection ratio may have partly hidden the positive effects of the clonal mixture.
Image‐guided spinal injection for diagnosis and therapy
Authors: Galloway Howard R
Published: August 2003

Over the past decade, major advances have been made in our ability to identify a putative cause of patients’ spinal pain using image‐guided injection techniques. These techniques might also provide a means of temporarily relieving patients’ pain and facilitating increased mobility. This article aims to review the rationale for and the techniques of the more commonly used spinal injection techniques.
Emergency embolization of spontaneous ruptured hepatocellular carcinoma: Correlation between survival and Child−Pugh classification
Authors: Lau KY , Wong TP , Wong WWC , Tan LTH , Chan JKW , Lee ASL
Published: August 2003

From February 1994 to April 2000, 29 emergency gelfoam embolizations for spontaneous ruptured hepatocellular carcinoma (HCC) performed in 28 patients were retrospectively reviewed. There were 11 patients in Child's A , 11 in Child's B and six in Child's C classification of cirrhosis. The duration of the procedure, artery embolized and complications were reviewed, and the Child−Pugh classification of each patient was correlated with their mean survival period. Embolization was done in 12 right hepatic arteries, two left hepatic arteries and 15 proper hepatic arteries. In one patient, the left hepatic artery was embolized initially but the proper hepatic artery was also embolized because another subcapsular liver tumour was found after reviewing the preangiogram CT scan. The entire procedure took 40−170 min (mean = 86 min) with no periprocedural complication. Following embolization, the mean survival period for Child's A class was 218.3 days, Child's B class was 83.4 days and Child's C class was 11.0 days. Transcatheter embolization is an effective treatment to arrest bleeding in spontaneous ruptured HCC. Patients with Child's A class cirrhosis have the longest survival. Selective embolization of either the right or the left hepatic artery alone carries the potential risk of missing multifocal HCC that might not be easily appreciated during angiography.
Precision of bone densitometry measurements: When is change true change and does it vary across bone density values?
Authors: Wong Joseph CH , Griffiths Matthew R
Published: August 2003

The precision error of the bone densitometer is used to interpret significant change in bone mineral density (BMD) in serial studies. The precision error can be expressed as standard deviation (SD) or coefficient of variation (CV). The aims of this study are to determine the precision error over a range of BMD values and to demonstrate the application of the precision error in clinical practice. A bone phantom was used consisting of a perspex block with eight compartments containing varying amounts of hydroxyapatite powder to simulate a range of bone densities. The block was scanned 21 times and manual regions placed over each compartment to measure the BMD in each compartment. There were no significant differences in the variances or SD for all eight compartments, that is, over the range of BMD normally encountered in clinical practice. However, the calculated CV show a progressive fall in values as the BMD rises. Therefore, the SD should be used to calculate significant BMD change. In a practise with quality control procedures in place to detect calibration drift and with appropriately trained personnel, a change of approximately 0.05 g/cm2 is generally regarded as being a significant change at a 95% confidence level.
Hydrodilatation in the management of shoulder capsulitis
Authors: Bell Simon , Coghlan Jennifer , Richardson Martin
Published: August 2003

The aim of this study was to research the benefit of hydraulic arthrographic capsular distension (hydrodilatation) in the management of adhesive capsulitis of the shoulder. One hundred and nine shoulders with primary adhesive capsulitis were treated with hydrodilatation. Prior to the procedure, 93 shoulders were painful. Two months following the procedure, 31 continued to have some pain. In the 109 shoulders, the measured range of passive glenohumeral movement improved by approximately 30° in all directions. The procedure was of similar benefit if carried out early or late in the disease process. The absolute improvement in movement range was similar in severe and mild cases. The severe cases in the long term, although improved, still had more restriction in movement and tended to have more pain than the other cases. There was considerable improvement in all the non‐diabetic patients. The patients with diabetes responded less well in the long term to hydrodilatation and had an increased requirement for arthroscopic surgery. Effective treatment of adhesive capsulitis can be achieved in the majority of cases with an immediate hydrodilatation of the shoulder. Technically, it is important to achieve maximum distension, preferably with capsular rupture, and to utilize cortisone in the fluid injected.
Triangular cord sign and ultrasound features of the gall bladder in infants with biliary atresia
Authors: Visrutaratna Pannee , Wongsawasdi Lumduan , Lerttumnongtum Pailin , Singhavejsakul Jesda , Kattipattanapong Vinaisak , Ukarapol Nuthapong
Published: August 2003

The aim of this study was to reassess the accuracy of the triangular cord sign, the triangular cord sign coupled with abnormal gall‐bladder length, and an irregular gall‐bladder wall in the diagnosis of biliary atresia. The ultrasonograms of 46 infants with cholestatic jaundice were reviewed for the triangular cord sign, gall‐bladder length and gall‐bladder wall without knowledge of the clinical data. Of the 23 infants with biliary atresia, 22 had the triangular cord sign whereas 17 infants with other causes of cholestatic jaundice did not have the triangular cord sign. The sensitivity, specificity, accuracy and positive predictive value of the triangular cord sign in the diagnosis of biliary atresia were 95.7, 73.9, 84.8 and 78.6%, respectively. The sensitivity, specificity, accuracy and positive predictive value of the triangular cord sign coupled with abnormal gall‐bladder length in the diagnosis of biliary atresia were all 95.7%. Gall‐bladder wall irregularity was seen in seven of 14 infants (50%) with biliary atresia whose gall bladders contained bile on ultrasound and in two of 22 infants (9.1%) without biliary atresia whose gall bladders contained bile on ultrasound. At the medical centre where this study was performed and where infants present with cholestatic jaundice at an advanced stage, the ultrasonographic triangular cord sign coupled with abnormal gall‐bladder length is more reliable than the ultrasonographic triangular cord sign alone or gall‐bladder wall irregularity in the diagnosis of biliary atresia.
Radiologists’ detection of mammographic abnormalities with and without a computer‐aided detection system
Authors: Quek ST , Thng CH , Khoo JBK , Koh WL
Published: August 2003

The aim of this study was to to evaluate the role of a computer‐aided program (CAD) in assisting detection of mammographic lesions by radiologists not specifically trained in mammography and its potential utility in breast screening. Mammograms were evaluated by radiologists not specifically trained in mammography first without, and then with, the CAD. Finally, the mammograms were evaluated by experienced mammographers who formed the reference standard. Two hundred and ninety four breasts were studied. In 257 breasts (87.4%), the CAD system did not help with the detection of abnormalities. It assisted radiologists in detecting abnormalities in 21 breasts (7.1%) with potential for detecting abnormalities in a further 13 breasts (4.4%). Only in three cases (1%) did it cause confusion in interpretation. There is overall increased sensitivity in detecting mammographic abnormalities with the aid of the CAD system from 74.4 to 87.2%, which is statistically significant. However, it failed to detect suspicious abnormalities in 71 breasts (24.1%). The CAD system improved detection of suspicious mammographic abnormalities by radiologists who are not specifically trained in mammography. However, there is also a substantial failure to detect suspicious mam­mographic features that cautions against over‐reliance on the system, emphasizing its role as a second reader at best.
Utility‐adjusted analysis of the cost of palliative radiotherapy for bone metastases
Authors: Barton Michael B , Jacob Susannah A , Gebsky Val
Published: August 2003

Palliative radiotherapy is effective in the treatment of bone metastases but is under‐utilized, possibly because it is perceived to be expensive. We performed a cost‐utility analysis of palliative radiotherapy for bone metastases, evaluating both the actual cost of radiotherapy as well as its impact on quality of life by adjusting for the variation in response to treatment. Hospital records between July 1991 and July 1996 were reviewed to ascertain the number of patients treated with palliative radiotherapy for bone metastases, the average number of fields of radiation delivered to each patient and the average duration of survival. Partial and complete response rates to palliative radiotherapy were obtained from a review of all published randomized controlled trials of radiation treatment of bone metastases. Utility values were assigned to the response rates, and an overall adjusted response rate to radiotherapy was derived. The cost of delivering a field of radiation was calculated. The total cost was divided by the total number of response months to give a utility‐adjusted cost per month of palliative radiotherapy. The utility‐adjusted cost per month of palliative radiotherapy of bone metastases was found to be AUS$ 100 per month or AUS$ 1200 per utility‐adjusted life‐year. This study demonstrates that, contrary to popular perception, palliative radiotherapy is a cost‐effective treatment modality for bone metastases.
Early cervical cancer treated with definitive or adjuvant radiotherapy: Improved survival with adjuvant radiotherapy attributable to patient selection
Authors: MacLeod Craig A , Vinod Shalini K
Published: August 2003

The optimum management of patients with early cervical cancer (Federation Internationale de Gynécologie Stages IB and IIA) remains controversial. We reviewed our radiotherapy practise and compared patients with early stage cervical cancer who had surgery and adjuvant radiotherapy (ART) to those that had definitive radiotherapy (DRT). One hundred and twenty‐seven patients were identified, 81 of these underwent ART and 46 underwent DRT. Patients who underwent DRT were significantly older and of poorer performance status than those who underwent ART. The 5‐year relapse‐free survival in the ART and DRT groups were 79 and 72%, respectively (P = 0.70). The corresponding 5‐year overall survival figures were 86 and 58% (P = 0.006). The difference was due to increased deaths from other causes in the DRT arm, 37 versus 7% (P = 0.0007.) The poorer overall survival of DRT patients was due to patient selection.
A prospective trial of short‐fractionation radiotherapy for the palliation of liver metastases
Authors: Bydder S , Spry NA , Christie DRH , Roos D , Burmeister BH , Krawitz H , Davis S , Joseph DJ , Poulsen M , Berry M
Published: August 2003

The purpose of this study was to prospectively examine the effectiveness and tolerability of a simple radiotherapy technique for the palliation of symptomatic liver metastases. Twenty‐eight patients with symptomatic liver metastases were enrolled from seven centres, and received targeted (partial or whole) liver irradiation consisting of 10 Gy in two fractions over 2 days. Symptoms at baseline were hepatic pain (27 patients), abdominal distension (19), night sweats (12), nausea (18) and vomiting (eight). Twenty‐two patients (76%) had failed previous treatment with chemotherapy, hormonal therapy and/or high‐dose steroids. Symptoms and potential toxicities were prospectively assessed at the time of treatment, then 2, 6 and 10 weeks later. Individual symptom response rates were 53−66% at 2 weeks. Partial or complete global symptomatic responses were noted in 15 patients (54%) overall. The treatment was well tolerated with two patients (7%) experiencing grade 3 toxicity (one vomiting and one diarrhoea); however, four patients reported temporary worsening of pain shortly after treatment. This simple and well‐tolerated treatment achieves useful palliation.
Multicentre dosimetric comparison of photon‐junctioning techniques in head and neck radiotherapy
Authors: Kron Tomas , Barnes Kara , O'Brien Peter
Published: August 2003

Because many head and neck radiotherapy treatment techniques rely on a junction between X‐ray fields, it was the aim of the present study to investigate the use of different junctioning techniques and the affect on the dose across the junction. Techniques in use at nine radiotherapy centres in Australia were investigated using thermoluminescence dosimetry (TLD). The techniques could broadly be divided into two groups: (i) use of the light field to match the fields after moving the patient; and (ii) use of asymmetric collimation to create a single isocentre located in the junction. The mean dose at the junction and its reproducibility was studied in five consecutive treatments in each centre using 25 TLD chips placed throughout the junction in an anthropomorphic phantom. There was a tendency for the mono‐isocentric technique to deliver a lower, more accurate mean dose at the junction (Group I: 1.22 Gy (n = 8) vs Group II: 0.96 Gy (n = 5) for 1 Gy planned, some centres contributed to both technique) with greater reproducibility (Group I: 9.6%, Group II: 5.1% of the mean dose). We conclude that a mono‐isocentric treatment technique has the potential to deliver a more accurate and reproducible dose distribution at the field junction of photon beams in head and neck treatment.