bluuebunny/crossref_metadata_2025 huggingface dataset:
User involvement and the NHS reforms
Authors: Rhodes Penny , Nocon Andrew
Published: March 2003

The policy of `user involvement' in the UK National Health Service emerged during the 1990s along with the reforms that created an internal market. Despite the official rhetoric, progress has been limited. Critics suggest that, not only was the policy flawed in its conception by the construction of service users as consumers and the conflation of consumerism with empowerment, but collaborative models of involvement have tended to legitimate rather than challenge existing provision. Some commentators have questioned the value of user involvement initiatives and proposed that alternative approaches, such as a strengthening of procedural rights or alignment with broader political campaigns, would be more appropriate. The low prominence given in the recent Government White Paper The New NHS1 to the contribution of service users, however, represents less of an ideological shift than a concentration on other, in the Government's view, more pressing priorities: namely, a concern to address the problems of public legitimacy and low staff morale by engaging in greater public participation and giving health professionals a more central role. The result has been a weakening of the users' voice by a conflation of user involvement with public participation and giving health professionals the authority to define users' needs for them. Service users risk, not only having their contribution devalued, but losing the right to an independent and distinctive voice. There is a real danger that the issues of user involvement will not be included on local agendas and the disparities between provision and need and between professionals' and users' views will increase.
Patient expectations and health‐related quality of life
Authors: Staniszewska Sophie
Published: March 2003

Objective

The measurement of health‐related quality of life (HRQL) has become increasingly common in health services research. Whilst useful, its focus on behaviour, capacities and activities means that it remains relatively specific. This paper explores the possibility of extending the evaluation of health by considering the concept of patients’ expectations.

Design

In‐depth and semi‐structured interviews were used to explore the concept of expectations from the patients’ perspective. Patients’ expectations were then used in the construction of a two‐part questionnaire.

Settings and participants

Expectations were explored with a group of 33 cardiac patients. The resulting questionnaire was given to 400 cardiac patients in a large teaching hospital in London.

Results

Patients identified a range of expectations which related to their health and seemed to represent the desired results of their hospital stay. Comparison of the content of patient expectations with a commonly used generic measure of HRQL, the Short‐Form 36 (SF‐36), found some overlap but indicated that patients seemed to adopt a broader approach to their health. Expectations that patients identified were used to construct two scales to measure expectations and their evaluation. The internal consistency of these scales was 0.82 and 0.88, respectively.

Conclusion

The study indicates the potential complexity of the concept of expectations and the need for further exploration. It also demonstrates the feasibility of constructing standardized scales to measure patient expectations. Whilst conceptually different from HRQL such standardized expectations scales could provide a useful adjunct to HRQL measurement and provide a meaningful context for the interpretation of HRQL data.
Postal surveys in primary care: the hidden burden
Authors: Jinks Clare , Ong Bie Nio , Croft Peter
Published: March 2003

Objective To investigate and analyse concerns raised by unsolicited mailing of a health survey to a community sample of older people.

Design Observation and monitoring of all telephone calls received throughout a 6‐week survey mailing period.

Setting and participants A total sample of all those aged over 50 years registered with three general practices in North Staffordshire (n=8995).

Main variable studied The frequency of telephone contact following the receipt of a postal questionnaire, and the nature of any associated distress.

Results and conclusions Individuals who receive unsolicited postal surveys may experience anxiety because of the actual receipt of the questionnaire, concerns about taking part or not taking part, personal issues, anger, worthiness or frustration with NHS services. Anxiety may also be triggered because of administrative issues, for example lost mail or overlap between mailing periods. Researchers can adopt measures both to reduce the potential anxiety and often hidden burden of postal surveys, and to facilitate individuals’ positive participation in research.
The DNA‐polymerase inhibiting activity of poly(β‐l‐malic acid) in nuclear extract during the cell cycle of Physarum polycephalum
Authors: Doerhoefer Sabine , Windisch Christina , Angerer Bernhard , Lavrik Olga I. , Lee Bong‐Seop , Holler Eggehard
Published: March 2003

The naturally synchronous plasmodia of myxomycetes synthesize poly(β‐l‐malic acid), which carries out cell‐specific functions. In Physarum polycephalum, poly(β‐l‐malate) [the salt form of poly(β‐l‐malic acid)] is highly concentrated in the nuclei, repressing DNA synthetic activity of DNA polymerases by the formation of reversible complexes. To test whether this inhibitory activity is cell‐cycle‐dependent, purified DNA polymerase α of P. polycephalum was added to the nuclear extract and the activity was measured by the incorporation of [3H]thymidine 5′‐monophosphate into acid precipitable nick‐activated salmon testis DNA. Maximum DNA synthesis by the reporter was measured in S‐phase, equivalent to a minimum of inhibitory activity. To test for the activity of endogenous DNA polymerases, DNA synthesis was followed by the highly sensitive photoaffinity labeling technique. Labeling was observed in S‐phase in agreement with the minimum of the inhibitory activity. The activity was constant throughout the cell cycle when the inhibition was neutralized by the addition of spermidine hydrochloride. Also, the concentration of poly(β‐l‐malate) did not vary with the phase of the cell cycle [Schmidt, A., Windisch, C. & Holler, E. (1996) Nuclear accumulation and homeostasis of the unusual polymer poly(β‐l‐malate) in plasmodia of Physarum polycephalum. Eur. J. Cell Biol.70, 373–380]. To explain the variation in the cell cycle, a periodic competition for poly(β‐l‐malate) between DNA polymerases and most likely certain histones was assumed. These␣effectors are synthesized in S‐phase. By competition␣they displace DNA polymerase from the complex of poly(β‐l‐malate). The free polymerases, which are no longer inhibited, engage in DNA synthesis. It is speculated that poly(β‐l‐malate) is active in maintaining mitotic synchrony of plasmodia by playing the mediator between the periodic synthesis of certain proteins and the catalytic competence of DNA polymerases.
The insert within the catalytic domain of tripeptidyl‐peptidase II is important for the formation of the active complex
Authors: Tomkinson Birgitta , Ní Laoi Bairbre , Wellington Kimberly
Published: March 2003

Tripeptidyl‐peptidase II (TPP II) is a large (Mr>106) tripeptide‐releasing enzyme with an active site of the subtilisin‐type. Compared with other subtilases, TPP II has a 200 amino‐acid insertion between the catalytic Asp44 and His264 residues, and is active as an oligomeric complex. This study demonstrates that the insert is important for the formation of the active high‐molecular mass complex. A recombinant human TPP II and a murine TPP II were found to display different complex‐forming characteristics when over‐expressed in human 293‐cells; the human enzyme was mainly in a nonassociated, inactive state whereas the murine enzyme formed active oligomers. This was surprising because native human TPP II is purified from erythrocytes as an active oligomeric complex, and the amino‐acid sequences of the human and murine enzymes were 96% identical. Using a combination of chimeras and a single point mutant, the amino acid responsible for this difference was identified as Arg252 in the recombinant human sequence, which corresponds to a glycine in the murine sequence. As Gly252 is conserved in all sequenced variants of TPP II, the recombinant enzyme with Arg252 is atypical. Nevertheless, as Arg252 evidently interferes with complex formation, and this residue is close to the catalytic His264, it may also explain why oligomerization influences enzyme activity. The exact mechanism for how the G252R substitution interferes with complex formation remains to be determined, but will be of importance for the understanding of the unique properties of TPP II.
CK2βtes gene encodes a testis‐specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster
Authors: Kalmykova Alla I. , Shevelyov Yuri Y. , Polesskaya Oksana O. , Dobritsa Anna A. , Evstafieva Alexandra G. , Boldyreff Brigitte , Issinger Olaf‐Georg , Gvozdev Vladimir A.
Published: March 2003

An earlier described CK2βtes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory β subunit of casein kinase 2. Western‐analysis using anti‐CK2βtes Ig revealed CK2βtes protein in Drosophila testes extract. Expression of a CK2βtes–β‐galactosidase fusion protein driven by the CK2βtes promoter was found in transgenic flies at postmitotic stages of spermatogenesis. Examination of biochemical characteristics of a recombinant CK2βtes protein expressed in Escherichia coli revealed properties similar to those of CK2β: (a) CK2βtes protein stimulates CK2α catalytic activity toward synthetic peptide; (b) it inhibits phosphorylation of calmodulin and mediates stimulation of CK2α by polylysine; (c) it is able to form (CK2βtes)2 dimers, as well as (CK2α)2(CK2βtes)2 tetramers. Using the yeast two‐hybrid system and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2βtes and CK2α. Northern‐analysis has shown that another regulatory (β′) subunit found recently in D. melanogaster genome is also testis‐specific. Thus, we describe the first example of two tissue‐specific regulatory subunits of CK2 which might serve to provide CK2 substrate recognition during spermatogenesis.
Artocarpus hirsuta lectin
Authors: Gaikwad Sushama M. , Gurjar Madhura M. , Khan M. Islam
Published: March 2003

Unfolding, inactivation and dissociation of the lectin from Artocarpus hirsuta seeds were studied by chemical (guanidine hydrochloride, GdnHCl) and thermal denaturation. Conformational transitions were monitored by intrinsic fluorescence and circular dichroism. The gradual red shift in the emission maxima of the native protein from 335 to 356 nm, change in the ellipticity at 218 nm and simultaneous decrease in the sugar binding activity were observed with increasing concentration of GdnHCl in the pH range between 4.0 and 9.0. The unfolding and inactivation by GdnHCl were partially reversible. Gel filtration of the lectin in presence of 1–6␣m GdnHCl showed that the protein dissociates reversibly into partially unfolded dimer and then irreversibly into unfolded inactive monomer. Thermal denaturation was irreversible. The lectin loses activity rapidly above 45 °C. The exposure of hydrophobic patches, distorted secondary structure and formation of insoluble aggregates of the thermally inactivated protein probably leads to the irreversible denaturation.
Dietary bisphenol A prevents ovarian degeneration and bone loss in female mice lacking the aromatase gene (Cyp19 )
Authors: Toda Katsumi , Miyaura Chisato , Okada Teruhiko , Shizuta Yutaka
Published: March 2003

We previously generated mice lacking aromatase activity by targeted disruption of Cyp19 (ArKO mice), and reported phenotypes of the female mice, showing hemorrhage formation and follicular depletion in the ovary, diminution in uterine size, and bone loss. In the present study, we examined the influence of dietary bisphenol A (BPA), a monomer used for the production of polycarbonate and known to have estrogenic activity, on these phenotypes of the ArKO mice. When ArKO mice were fed chow diets supplemented with 0.1% or 1% (w/w) BPA for 5 months, they were protected from ovarian degeneration, uterine diminution and bone loss in a dose‐dependent manner. Northern blot analyses of ovarian RNA of ArKO mice showed differences in the expression levels of insulin‐like growth factor (IGF)‐I, IGF‐I receptor, growth differentiation factor 9 and bone morphogenetic protein 15 as compared with those in the ovaries of wild‐type mice. The differences in the expression levels were restored by dietary BPA. In the ArKO uteri, expression of progesterone receptor and vascular endothelial growth factor mRNAs was diminished, and was restored by BPA to the levels in wild‐type mice. In contrast, BPA had little effect on the ovarian, uterine and skeletal structures of wild‐type mice. In conclusion, estrogenic effects of BPA on the reproductive tract as well as skeletal tissue were evident in adult female ArKO mice. These results suggest that the ArKO mouse is an animal model suitable for studying effects of estrogenic chemicals as well as estrogen in vivo.
Human immunoglobulin A (IgA)‐specific ligands from combinatorial engineering of protein A
Authors: Rönnmark Jenny , Grönlund Hans , Uhlén Mathias , Nygren Per‐Åke
Published: March 2003

Affinity reagents capable of selective recognition of the different human immunoglobulin isotypes are important detection and purification tools in biotechnology. Here we describe the development and characterization of affinity proteins (affibodies) showing selective binding to human IgA. From protein libraries constructed by combinatorial mutagenesis of a 58‐amino‐acid, three‐helix bundle domain derived from the IgG‐binding staphylococcal protein A, variants showing IgA binding were selected by using phage display technology and IgA monoclonal antibodies (myeloma) as target molecules. Characterization of selected clones by biosensor technology showed that five out of eight investigated affibody variants were capable of IgA binding, with dissociation constants (Kd) in the range between 0.5 and 3 µm. One variant (ZIgA1) showing the strongest binding affinity was further analyzed, and showed that human IgA subclasses (IgA1 and IgA2) as well as secretory IgA were recognized with similar efficiencies. No detectable cross‐reactivity towards human IgG, IgM, IgD or IgE was observed. The potential use of the ZIgA1 affibody as a ligand in affinity chromatography applications was first demonstrated by selective recovery of IgA protein from a spiked Escherichia coli total cell lysate, using an affinity column containing a divalent head‐to‐tail ZIgA1 affibody dimer construct as a ligand. In addition, efficient affinity recovery of IgA from unconditioned human plasma was also demonstrated.
Two 1 : 1 binding modes for distamycin in the minor groove of d(GGCCAATTGG)
Authors: Uytterhoeven Koen , Sponer Jiri , Van Meervelt Luc
Published: March 2003

Single‐crystal X‐ray structure determinations of the complex between the minor‐groove binder distamycin and d(GGCCAATTGG) reveal two 1 : 1 binding modes which differ in the orientation of the drug molecule in the minor groove. The two crystals were grown from different crystallization conditions and found to diffract to 2.38 and 1.85 Å, respectively. The structures were refined to completion using SHELXL‐93, resulting in a residual R factor of 20.30% for the 2.38‐Å resolution structure (including 46 water molecules) and 19.74% for the 1.85‐Å resolution structure (including 74 water molecules). In both orientations, bifurcated hydrogen bonds are formed between the amide nitrogen atoms of the drug and AT base pairs. With a binding site of at least five base pairs, close contacts between the terminal distamycin atoms and guanine amino groups are inevitable. The detailed nature of several of these interactions was further investigated by ab initio quantum chemical methods.
The Fc receptor γ‐chain is necessary and sufficient to initiate signalling through glycoprotein VI in transfected cells by the snake C‐type lectin, convulxin
Authors: Berlanga Oscar , Tulasne David , Bori Teresa , Snell Daniel C. , Miura Yoshiki , Jung Stephanie , Moroi Masaaki , Frampton Jonathan , Watson Steve P.
Published: March 2003

There is extensive evidence that FcR γ‐chain couples to the collagen receptor glycoprotein VI (GPVI) and becomes phosphorylated on tyrosines upon receptor cross‐linking. However, it is not established whether this receptor complex is sufficient to initiate the signalling cascade. We transfected GPVI and the FcR γ‐chain into the human erythroleukaemia cell line K562, which lacks detectable expression of GPVI and the FcR γ‐chain. The results show that GPVI is unable to signal when expressed alone, despite its surface expression, upon stimulation with the snake C‐type lectin, convulxin. Coexpression of the FcR γ‐chain confers signalling properties on the receptor. Furthermore, cotransfection of the FcR γ‐chain and two mutant versions of GPVI shows that the transmembrane arginine and cytoplasmic tail of GPVI are necessary for association with the FcR γ‐chain. These results demonstrate that reconstitution of the GPVI–FcR γ‐chain complex in cells expressing the necessary signalling network is sufficient to initiate signalling events in response to convulxin and collagen‐related peptide.
Trehalose‐based oligosaccharides isolated from the cytoplasm of Mycobacterium smegmatis
Authors: Ohta Masaya , Pan Y. T. , Laine Roger A. , D. Elbein Alan
Published: March 2003

A series of trehalose‐based oligosaccharides were isolated from the cytoplasmic fraction of Mycobacterium smegmatis and purified by gel‐filtration and paper chromatography and TLC. Their structures were determined by HPLC and GLC to determine sugar composition and ratios, MALDI‐TOF MS to measure molecular mass, methylation analysis to determine linkages, 1H‐NMR to obtain anomeric configurations of glycosidic linkages, and exoglycosidase digestions followed by TLC to determine sequences and anomeric configurations of the monosaccharides. Six different oligosaccharides were identified all with trehalose as the basic structure and additional glucose or galactose residues attached in various linkages. One of these oligosaccharides is the disaccharide trehalose (Glcα1–1αGlc), which is present in substantial amounts in these cells and also in other mycobacteria. Two other oligosaccharides, the tetrasaccharides Glcα1–4Glcα1–1αGlc6–1αGal and Galα1–6Galα1–6Glcα1–1αGlc, have not previously been isolated from natural sources or synthesized chemically. The fourth oligosaccharide, Glcβ1–6Glcβ1–6Glcα1–1αGlc, has been isolated from corynebacteria, but not reported in other organisms. Two other oligosaccharides, Glcα1–4Glcα1–1αGlc, which has been synthesized chemically and isolated from insects but not previously reported in mycobacteria, and Glcβ1–6Glcα1–1αGlc, which was previously isolated from Mycobacterium fortuitum and yeast, were also characterized. Another trisaccharide found in the cytosol has been partially characterized as arabinosyl‐1–4trehalose, but neither the anomeric configuration nor the d or l configuration of the arabinose is known. In analogy with sucrose and its higher homologs, raffinose and stachyose, which may act as protective agents during maturation drying in plants, these trehalose homologs may also have a protective role in mycobacteria, perhaps during latency.
Mutations in the docking site for cytochrome c on the Paracoccus heme aa3 oxidase
Authors: Drosou Viktoria , Malatesta Francesco , Ludwig Bernd
Published: March 2003

Introducing site‐directed mutations in surface‐exposed residues of subunit II of the heme aa3 cytochrome c oxidase of Paracoccus denitrificans, we analyze the kinetic parameters of electron transfer from reduced horse heart cytochrome c. Specifically we address the following issues: (a) which residues on oxidase contribute to the docking site for cytochrome c, (b) is an aromatic side chain required for electron entry from cytochrome c, and (c) what is the molecular basis for the previously observed biphasic reaction kinetics. From our data we conclude that tryptophan 121 on subunit II is the sole entry point for electrons on their way to the CuA center and that its precise spatial arrangement, but not its aromatic nature, is a prerequisite for efficient electron transfer. With different reaction partners and experimental conditions, biphasicity can always be induced and is critically dependent on the ionic strength during the reaction. For an alternative explanation to account for this phenomenon, we find no evidence for a second cytochrome c binding site on oxidase.
Dissecting the effect of trifluoroethanol on ribonuclease A
Authors: Köditz Jens , Arnold Ulrich , Ulbrich‐Hofmann Renate
Published: March 2003

With the aim to distinguish between local and global conformational changes induced by trifluoroethanol in RNase A, spectroscopic and activity measurements in combination with proteolysis by unspecific proteases have been exploited for probing structural transitions of RNase A as a function of trifluoroethanol concentration. At > 30% (v/v) trifluoroethanol (pH 8.0; 25 °C), circular dichroism and fluorescence spectroscopy indicate a cooperative collapse of the tertiary structure of RNase A coinciding with the loss of its enzymatic activity. In contrast to the denaturation by guanidine hydrochloride, urea or temperature, the breakdown of the tertiary structure in trifluoroethanol is accompanied by an induction of secondary structure as detected by far‐UV circular dichroism spectroscopy. Proteolysis with the nonspecific proteases subtilisin Carlsberg or proteinase K, both of which attack native RNase A at the Ala20‐Ser21 peptide bond, yields refined information on conformational changes, particularly in the pretransition region. While trifluoroethanol at concentrations > 40% results in a strong increase of the rate of proteolysis and new primary cleavage sites (Tyr76‐Ser77, Met79‐Ser80) were identified, the rate of proteolysis at trifluoroethanol concentrations < 40% (v/v) is much smaller (up to two orders of magnitude) than that of the native RNase A. The proteolysis data point to a decreased flexibility in the surrounding of the Ala20‐Ser21 peptide bond, which we attribute to subtle conformational changes of the ribonuclease A molecule. These changes, however, are too marginal to alter the overall catalytic and spectroscopic properties of ribonuclease A.
Redox reaction between amino‐(3,4‐dihydroxyphenyl)methyl phosphonic acid and dopaquinone is responsible for the apparent inhibitory effect on tyrosinase
Authors: Gąsowska Beata , Wojtasek Hubert , Hurek Józef , Drąg Marcin , Nowak Kornel , Kafarski Paweł
Published: March 2003

Amino‐(3,4‐dihydroxyphenyl)methyl phosphonic acid, the phosphonic analog of 3,4‐dihydroxyphenylglycine, had been previously reported as a potent inhibitor of tyrosinase. The mechanism of the apparent enzyme inhibition by this compound has now been established. Amino‐(3,4‐dihydroxyphenyl)methyl phosphonic acid turned out to be a substrate and was oxidized to o‐quinone, which evolved to a final product identified as 3,4‐dihydroxybenzaldehyde, the same as for 3,4‐dihydroxyphenylglycine. Monohydroxylated compounds (amino‐(3‐hydroxyphenyl)methyl phosphonic acid and amino‐(4‐hydroxyphenyl)methyl phosphonic acid) were not oxidized, neither was 4‐hydroxy‐l‐phenylglycine. However, the relatively high Km for amino‐(3,4‐dihydroxyphenyl)methyl phosphonic acid (0.52 mm) indicated that competitive inhibition could not entirely explain the previously reported strong inhibitory effect (Ki = 50 and 97 µm for tyrosine and 3‐(3,4‐dihydroxyphenyl)alanine (Dopa) as substrates, respectively). Neither was the enzyme covalently inactivated to a significant degree. Spectroscopic and electrochemical analysis of the oxidation of a mixture of Dopa and the inhibitor demonstrated that the phosphonic compound reduced dopaquinone back to Dopa, thus diminishing and delaying the formation of dopachrome. This produces an apparent strong inhibitory effect when the reaction is monitored spectrophotometrically at 475 nm. In this peculiar case Dopa acts as a redox shuttle mediating the oxidation of the shorter phosphonic homolog. Decomposition of the phosphonic o‐quinone to 3,4‐dihydroxybenzaldehyde drives the reaction against the slightly unfavorable difference in redox potentials.
Medium‐chain dehydrogenases/reductases (MDR)
Authors: Nordling Erik , Jörnvall Hans , Persson Bengt
Published: March 2003

Completed eukaryotic genomes were screened for medium‐chain dehydrogenases/reductases (MDR). In the human genome, 23 MDR forms were found, a number that probably will increase, because the genome is not yet fully interpreted. Partial sequences already indicate that at least three further members exist. Within the MDR superfamily, at least eight families were distinguished. Three families are formed by dimeric alcohol dehydrogenases (ADH; originally detected in animals/plants), cinnamyl alcohol dehydrogenases (originally detected in plants) and tetrameric alcohol dehydrogenases (originally detected in yeast). Three further families are centred around forms initially detected as mitochondrial respiratory function proteins, acetyl‐CoA reductases of fatty acid synthases, and leukotriene B4 dehydrogenases. The two remaining families with polyol dehydrogenases (originally detected as sorbitol dehydrogenase) and quinone reductases (originally detected as ζ‐crystallin) are also distinct but with variable sequences. The most abundant families in the human genome are the dimeric ADH forms and the quinone oxidoreductases. The eukaryotic patterns are different from those of Escherichia coli. Thedifferent families were further evaluated by molecular modelling of their active sites as to geometry, hydrophobicity and volume of substrate‐binding pockets. Finally, sequence patterns were derived that are diagnostic for the different families and can be used in genome annotations.
Inhibition of human MDA‐MB‐231 breast cancer cell invasion by matrix metalloproteinase 3 involves degradation of plasminogen
Authors: Farina Antonietta R. , Tacconelli Antonella , Cappabianca Lucia , Gulino Alberto , Mackay Andrew R
Published: March 2003

Matrix metalloproteinase (MMP)‐3 inhibited human MDA‐MB‐231 breast cancer cell invasion through reconstituted basement membranein vitro. Inhibition of invasion was dependent upon plasminogen and MMP‐3 activation, was impaired by the peptide MMP‐3 inhibitor Ac‐Arg‐Cys‐Gly‐Val‐Pro‐Asp‐NH2and was associated with: rapid MMP‐3‐mediated plasminogen degradation to microplasminogen and angiostatin‐like fragments; the removal of single‐chain urokinase plasminogen activator from MDA‐MB‐231 cell membranes; impaired membrane plasminogen association; reduced rate of tissue plasminogen activator (t‐PA) and membrane‐mediated plasminogen activation; and reduced laminin‐degrading capacity. Purified human plasminogen lysine binding site‐1 (kringles 1–3) exhibited a similar capacity to inhibit MDA‐MB‐231 invasion, impair t‐PA and cell membrane‐mediated plasminogen activation and impair laminin degradation by plasmin. Our data provide evidence that MMP‐3 can inhibit breast tumour cell invasionin vitroby a mechanism involving plasminogen degradation to fragments that limit plasminogen activation and the degradation of laminin. This supports the hypothesis that MMP‐3, under certain conditions, may protect against tumour invasion, which would help to explain why MMP‐3 expression, associated with benign and early stage breast tumours, is frequently lost in advanced stage, aggressive, breast disease.
Solution NMR structure of five representative glycosylated polyene macrolide antibiotics with a sterol‐dependent antifungal activity
Authors: Volpon Laurent , Lancelin Jean‐Marc
Published: March 2003

Glycosylated polyene macrolide antibiotics, as nystatins and amphotericins, are amphiphilic structures known to exert antifungal activity by disrupting the fungal cell membrane, leading to leakage of cellular materials, and cell death. This membrane disruption is strongly influenced by the presence and the exact nature of the membrane sterols. The solution structures of five representative glycosylated members, three tetraenes (pimaricin, nystatin A1 and rimocidin) and two heptaenes (candidin and vacidin A) have been calculated using geometric restraints derived from 1H‐NMR data and random searches of their conformational space. Despite a different apparent structural order, the NMR solutions structure indicate that the hydroxyl groups all clustered on one side of the rod‐shaped structures, and the glycosyl moieties are structurally conserved both in their conformation and their apparent order. The molecular structures afford an understanding of their selective interaction with the membrane sterols and the design of new polyene macrolides with improved activities.
Antibacterial and antifungal properties of α‐helical, cationic peptides in the venom of scorpions from southern Africa
Authors: Moerman Leentje , Bosteels Suzanne , Noppe Wim , Willems Jean , Clynen Elke , Schoofs Liliane , Thevissen Karin , Tytgat Jan , Van Eldere Johan , van der Walt Jurg , Verdonck Fons
Published: March 2003

Two novel pore‐forming peptides have been isolated from the venom of the South‐African scorpion Opistophtalmus carinatus. These peptides, designated opistoporin 1 and 2, differ by only one amino acid and belong to a group of α‐helical, cationic peptides. For the first time, a comparison of the primary structures of α‐helical pore‐forming peptides from scorpion venom was undertaken. This analysis revealed that peptides in the range of 40–50 amino acids contain a typical scorpion conserved sequence S(x)3KxWxS(x)5L. An extensive study of biological activity of synthesized opistoporin 1 and parabutoporin, a pore‐forming peptide previously isolated from the venom of the South‐African scorpion Parabuthus schlechteri, was undertaken to investigate an eventual cell‐selective effect of the peptides. Opistoporin 1 and parabutoporin were most active in inhibiting growth of Gram‐negative bacteria (1.3–25 µm), while melittin and mastoparan, two well‐known cytolytic peptides, were more effective against Gram‐positive bacteria in the same concentration range. In addition, the peptides showed synergistic activity with some antibiotics commonly used in therapy. Opistoporin 1 and parabutoporin had hemolytic activity intermediate between the least potent mastoparan and the highly lytic melittin. Furthermore, all peptides inhibited growth of fungi. Experiments with SYTOX green suggested that this effect is related to membrane permeabilization.
Intracellular localization and transcriptional regulation of tumor necrosis factor (TNF) receptor‐associated factor 4 (TRAF4)
Authors: Glauner Heike , Siegmund Daniela , Motejadded Hassan , Scheurich Peter , Henkler Frank , Janssen Ottmar , Wajant Harald
Published: March 2003

To gain insight in the subcellular localization of tumor necrosis factor receptor‐associated factor (TRAF4) we analyzed GFP chimeras of full‐length TRAF4 and various deletion mutants derived thereof. While TRAF4–GFP (T4–GFP) was clearly localized in the cytoplasm, the N‐terminal deletion mutant, T4(259–470), comprising the TRAF domain of the molecule, and a C‐terminal deletion mutant consisting mainly of the RING and zinc finger domains of TRAF4 were both localized predominantly to the nucleus. Passive nuclear localization of T4(259–470) can be ruled out as the TRAF domain of TRAF4 was sufficient to form high molecular weight complexes. T4(259–470) recruited full‐length TRAF4 into the nucleus whereas TRAF4 was unable to change the nuclear localization of T4(259–470). Thus, it seems that individual T4(259–470) mutant molecules are sufficient to direct the respective TRAF4–T4(259–470) heteromeric complexes into the nucleus. In cells forming cell–cell contacts, TRAF4 was recruited to the sites of contact via its C‐TRAF domain. The expression of some TRAF proteins is regulated by the NF‐κB pathway. Thus, we investigated whether this pathway is also involved in the regulation of the TRAF4 gene. Indeed, in primary T‐cells and Jurkat cells stimulated with the NF‐κB inducers TNF or phorbol 12‐myristate 13‐acetate (PMA), TRAF4‐mRNA was rapidly up‐regulated. In Jurkat T‐cells deficient for I‐κB kinase γ (IKKγ, also known as NEMO), an essential component of the NF‐κB‐inducing–IKK complex, induction of TRAF4 was completely inhibited. In cells deficient for RIP (receptor interactive protein), an essential signaling intermediate of TNF‐dependent NF‐κB activation, TNF‐, but not PMA‐induced up‐regulation of TRAF4 was blocked. These data suggest that activation of the NF‐κB pathway is involved in up‐regulation of TRAF4 in T‐cells.
Two different E2F6 proteins generated by alternative splicing and internal translation initiation
Authors: Dahme Tillman , Wood Jason , Livingston David M. , Gaubatz Stefan
Published: March 2003

E2F transcription factors play an important role in the regulation of cell cycle progression. E2F6, the most recently identified member of the E2F family, is a retinoblastoma‐protein‐independent transcriptional repressor that is required for developmental patterning of the axial skeleton. It has recently been shown that the E2f6 locus produces two different mRNAs, E2F6 and E2F6b. The E2F6b mRNA contains an additional exon that is inserted by alternative splicing. This exon contains an in‐frame stop‐codon and an in‐frame translation initiation codon. However, whether a protein is translated from the E2F6b mRNA has not yet been addressed. We now show that internal translation initiation gives rise to E2F6b, an amino‐terminal truncated E2F6 protein. We also show that E2F6 and E2F6b mRNAs are ubiquitously expressed in primary mouse tissues. During the cell cycle, the highest expression of both forms is found at the G1 to S transition. The 5′ untranslated regions of E2F6 and E2F6b are unusually long, and they contain several upstream AUG codons followed by short reading frames. Our results suggest that translation of E2F6b is initiated by internal ribosome entry. We propose that regulated translation initiation can produce distinct E2F6 isoforms under different physiological conditions.
Ascidian arrestin (Ci‐arr), the origin of the visual and nonvisual arrestins of vertebrate
Authors: Nakagawa Masashi , Orii Hidefumi , Yoshida Norihiro , Jojima Eri , Horie Takeo , Yoshida Reiko , Haga Tatsuya , Tsuda Motoyuki
Published: March 2003

Arrestin is one of the key proteins for the termination of G protein signaling. Activated G protein‐coupled receptors (GPCRs) are specifically phosphorylated by G protein‐coupled receptor kinases (GRKs) and then bind to arrestins to preclude the receptor/G protein interaction, resulting in quenching of the following signal transduction. Vertebrates possess two types of arrestin; visual arrestin expressed exclusively in photoreceptor cells in retinae and pineal organs, and β‐arrestin, which is expressed ubiquitously. Unlike visual arrestin, β‐arrestin contains the clathrin‐binding domain at the C‐terminus, responsible for the agonist‐induced internalization of GPCRs. Here, we isolated a novel arrestin gene (Ci‐arr) from the primitive chordate, the ascidian Ciona intestinalis larvae. The deduced amino acid sequence suggests that Ci‐Arr be closely related to vertebrate arrestins. Interestingly, this arrestin has the feature of both visual and β‐arrestin. Whereas the expression of Ci‐arr was restricted to the photoreceptors in the larvae similarly to visual arrestin, the gene product, containing the clathrin‐binding domain, promoted the GPCR internalization in HEK293tsA201 cells similarly to β‐arrestin. The phylogenetic tree shows that Ci‐Arr is branched from a common root of visual and β‐arrestins. Southern analysis suggests that the Ciona genome contains only one gene for the arrestin family. These results suggest that the visual and β‐arrestin genes were generated by the duplication of the prototypical arrestin gene like Ci‐arr in the early evolution of vertebrates.
Development of a selective photoactivatable antagonist for corticotropin‐releasing factor receptor, type 2 (CRF2)
Authors: Bonk Ines , Rühmann Andreas
Published: March 2003

A novel photoactivatable analog of antisauvagine‐30 (aSvg‐30), a specific antagonist for corticotropin‐releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. The N‐terminal amino‐acid d‐Phe in aSvg‐30 [d‐Phe11,His12]Svg(11−40) was replaced by a phenyldiazirine, the 4‐(1‐azi‐2,2,2‐trifluoroethyl)benzoyl (ATB) residue. The photoactivatable aSvg‐30 analog ATB‐[His12]Svg was tested for its ability to displace [125I‐Tyr0]oCRF or [125I‐Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRF1) or mouse CRF receptor, type 2β (mCRF2β). Furthermore, the ability of ATB‐[His12]Svg(12−40) to inhibit oCRF‐ or Svg‐stimulated cAMP production of transfected HEK 293 cells expressing either rCRF1 (HEK‐rCRF1 cells) or mCRF2β (HEK‐mCRF2β cells) was determined. Unlike astressin and photo astressin, ATB‐[His12]Svg(12−40) showed high selective binding to mCRF2β (Ki = 3.1 ± 0.2 nm) but not the rCRF1 receptor (Ki = 142.5 ± 22.3 nm) and decreased Svg‐stimulated cAMP activity in mCRF2β‐expressing cells in a similar fashion as aSvg‐30. A 66‐kDa protein was identified by SDS/PAGE, when the radioactively iodinated analog of ATB‐[His12]Svg(12−40) was covalently linked to mCRF2β receptor. The specificity of the photoactivatable 125I‐labeled CRF2β antagonist was demonstrated with SDS/PAGE by the finding that this analog could be displaced from the receptor by antisauvagine‐30, but not other unrelated peptides such as vasoactive intestinal peptide (VIP).
Baboon cytochrome P450 17α‐hydroxylase/17,20‐lyase (CYP17)
Authors: Swart Amanda C. , Kolar Norbert W. , Lombard Nic , Mason J. Ian , Swart Pieter
Published: March 2003

Human cytochrome P450 17α‐hydroxylase (CYP17) catalyses not only the 17α‐hydroxlation of pregnenolone and progesterone and the C17,20‐side chain cleavage (lyase) of 17α‐hydroxypregnenolone, necessary for the biosynthesis of C21‐glucocorticoids and C19‐androgens, but also catalyses the 16α‐hydroxylation of progesterone. In efforts to understand the complex enzymology of CYP17, structure/function relationships have been reported previously after expressing recombinant DNAs, encoding CYP17 from various species, in nonsteroidogenic mammalian or yeast cells. A major difference between species resides in the lyase activity towards the hydroxylated intermediates and in the fact that the secretion of C19‐steroids take place, in some species, principally in the gonads. Because human and higher primate adrenals secrete steroids, CYP17 has been characterized in the Cape baboon, a species more closely related to humans, in an effort to gain a further understanding of the reactions catalysed by CYP17. Baboon and human CYP17 cDNA share 96% homology. Baboon CYP17 has apparent Km and V values for pregnenolone and progesterone of 0.9 µm and 0.4 nmol·h−1·mg protein−1 and 6.5 µm and 3.9 nmol·h−1·mg protein−1, respectively. Baboon CYP17 had a significantly higher activity for progesterone hydroxylation relative to pregnenolone. No 16α‐hydroxylase and no lyase activity for 17α‐hydroxyprogesterone. Sequence analyses showed that there are 28 different amino acid residues between human and baboon CYP17, primarily in helices F and G and the F‐G loop.
Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoides
Authors: Sauvageot Nicolas , Pichereau Vianney , Louarme Loïc , Hartke Axel , Auffray Yanick , Laplace Jean‐Marie
Published: March 2003

The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpression in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homogeneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively. They were identified by mass spectrometry to correspond to the large, medium and small subunits of the dehydratase encoded by the pduC, pduD and pduE genes, respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC, D, E genes (61, 24.7 and 19,1 kDa, respectively) and a α2β2γ2 composition. The Km for the three main substrates were 1.6 mm for 1,2‐propanediol, 5.5 mm for 1,2‐ethanediol and 8.3 mm for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 µm. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 °C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme, another protein involved in the 1,2‐propanediol metabolism pathway.
RING finger, B‐box, and coiled‐coil (RBCC) protein expression in branchial epithelial cells of Japanese eel, Anguilla japonica
Authors: Miyamoto Kentaro , Nakamura Nobuhiro , Kashiwagi Masahide , Honda Shinji , Kato Akira , Hasegawa Sanae , Takei Yoshio , Hirose Shigehisa
Published: March 2003

An RBCC (RING finger, B‐box, and coiled‐coil) protein was identified that belongs to the superfamily of zinc‐binding proteins and is specifically expressed in the gill of eel, Anguilla japonica. Euryhaline fishes such as eels can migrate between freshwater and seawater, which is considered to be accomplished by efficient remodeling of the architecture and function of the gill, a major osmoregulatory organ. To identify molecules involved in such adaptive changes, we performed differential display using mRNA preparations from freshwater and seawater eel gills and obtained an RBCC clone among several differentially expressed clones. The clone encoded a protein of 514 amino acid residues with structural features characteristic of the RBCC protein; we therefore named it eRBCC (e for eel). eRBCC mRNA was specifically expressed in the gills with a greater extent in the gills of freshwater eels. Immunohistochemistry revealed that the expression of eRBCC is confined to particular epithelial cells of the gills including freshwater‐specific lamellar chloride cells. The RING finger of eRBCC was found to have a ubiquitin ligase activity, suggesting an important regulatory role of eRBCC in the remodeling of branchial cells.
Endogenous mono‐ADP‐ribosylation mediates smooth muscle cell proliferation and migration via protein kinase N‐dependent induction of c‐fos expression
Authors: Yau Lorraine , Litchie Brenda , Thomas Shawn , Storie Benjamin , Yurkova Natalia , Zahradka Peter
Published: March 2003

ADP‐ribosylation has been coupled to intracellular events associated with smooth muscle cell vasoreactivity, cytoskeletal integrity and free radical damage. Additionally, there is evidence that ADP‐ribosylation is required for smooth muscle cell proliferation. Our investigation employed selective inhibitors to establish that mono‐ADP‐ribosylation and not poly(ADP‐ribosyl)ation was necessary for the stimulation of DNA synthesis by mitogens. Mitogen treatment increased concomitantly the activity of both soluble and particulate mono‐ADP‐ribosyltransferase, as well as the number of modified proteins. Inclusion of meta‐iodobenzylguanidine (MIBG), a selective decoy substrate of arginine‐dependent mono‐ADP‐ribosylation, prevented the modification of these proteins. MIBG also blocked the stimulation of DNA and RNA synthesis, prevented smooth muscle cell migration and suppressed the induction of c‐fos and c‐myc gene expression. An examination of relevant signal transduction pathways showed that MIBG did not interfere with MAP kinase and phosphatidylinositol 3‐kinase stimulation; however, it did inhibit phosphorylation of the Rho effector, PRK1/2. This novel observation suggests that mono‐ADP‐ribosylation participates in a Rho‐ dependent signalling pathway that is required for immediate early gene expression.
Effects of various N‐terminal addressing signals on sorting and folding of mammalian CYP11A1 in yeast mitochondria
Authors: Kovaleva Irina E. , Novikova Lyudmila A. , Nazarov Pavel A. , Grivennikov Sergei I. , Luzikov Valentin N.
Published: March 2003

Topogenesis of cytochrome P450scc, a resident protein of the inner membrane of adrenocortical mitochondria, is still obscure. In particular, little is known about the cause of its tissue specificity. In an attempt to clarify this point, we examined the process in Saccharomyces cerevisiae cells synthesizing cytochrome P450scc as its native precursor (pCYP11A1) or versions in which its N‐terminal addressing presequence had been replaced with those of yeast mitochondrial proteins: CoxIV(1–25) and Su9(1–112). We found the pCYP11A1 and CoxIV(1–25)‐mCYP11A1 versions to be effectively imported into yeast mitochondria and subjected to proteolytic processing. However, only minor portions of the imported proteins were incorporated into mitochondrial membranes, whereas their bulk accumulated as aggregates insoluble in 1% Triton X‐100. Along with previously published data, this suggests that a distinguishing feature of the import of the CYP11A1 precursors into yeast mitochondria is their easy translocation into the matrix where the foreign proteins mainly undergo proteolysis or aggregation. The fraction of CYP11A1 that happens to be inserted into the inner mitochondrial membrane is effectively converted into the catalytically active holoenzyme. Experiments with the Su9(1–112)‐mCYP11A1 construct bearing a re‐export signal revealed that, after translocation of the fused protein into the matrix and its processing, the Su9(67–112) segment ensures association of the mCYP11A1 body with the inner membrane, but proper folding of the latter does not take place. Thus it can be said that the most specific stage of CYP11A1 topogenesis in adrenocortical mitochondria is its confinement and folding in the inner mitochondrial membrane. In yeast mitochondria, only an insignificant portion of the imported CYP11A1 follows this mechanism.
Functional characterization of Drosophila melanogaster PERK eukaryotic initiation factor 2α (eIF2α) kinase
Authors: Pomar Natalia , Berlanga Juan J. , Campuzano Sonsoles , Hernández Greco , Elías Mónica , de Haro César
Published: March 2003

Four distinct eukaryotic initiation factor 2α (eIF2α) kinases phosphorylate eIF2α at S51 and regulate protein synthesis in response to various environmental stresses. These are the hemin‐regulated inhibitor (HRI), the interferon‐inducible dsRNA‐dependent kinase (PKR), the endoplasmic reticulum (ER)‐resident kinase (PERK) and the GCN2 protein kinase. Whereas HRI and PKR appear to be restricted to mammalian cells, GCN2 and PERK seem to be widely distributed in eukaryotes. In this study, we have characterized the second eIF2α kinase found in Drosophila, a PERK homologue (DPERK).

Expression of DPERK is developmentally regulated. During embryogenesis, DPERK expression becomes concentrated in the endodermal cells of the gut and in the germ line precursor cells. Recombinant wild‐type DPERK, but not the inactive DPERK‐K671R mutant, exhibited an autokinase activity, specifically phosphorylated Drosophila eIF2α at S50, and functionally replaced the endogenous Saccharomyces cerevisiae GCN2. The full length protein, when expressed in 293T cells, located in the ER‐enriched fraction, and its subcellular localization changed with deletion of different N‐terminal fragments. Kinase activity assays with these DPERK deletion mutants suggested that DPERK localization facilitates its in vivo function. Similar to mammalian PERK, DPERK forms oligomers in vivo and DPERK activity appears to be regulated by ER stress. Furthermore, the stable complexes between wild‐type DPERK and DPERK‐K671R mutant were mediated through the N terminus of the proteins and exhibited an in vitro eIF2α kinase activity.