bluuebunny/crossref_metadata_2025 huggingface dataset:
Gelatin‐specific cellular immune responses persist for more than 3 years after priming with gelatin containing DTaP vaccine
Authors: Kumagai T. , Kamada M. , Igarashi C. , Yuri K. , Furukawa H. , Nagata N. , Saito A. , Okui T. , Yano S.
Published: March 2003

Background Gelatin‐specific cell‐mediated immunity develops in subjects inoculated with gelatin containing DTaP vaccine. However, it is not yet known whether such established sensitization to gelatin disappears or persists with time.

Objective The aim of this study was to follow the patients with gelatin sensitization elicited by DTaP vaccination for their lymphocyte responsiveness and IgE, IgG antibody specific to gelatin over several years and to compare the activities with those at the time of enrolment into the study.

Methods We studied 28 subjects who developed positive lymphocyte proliferation test (LPT) after receiving gelatin containing DTaP vaccine and eight subjects who had a negative LPT after inoculation of non‐gelatin DTaP. Determination of IgE, IgG antibodies and specific lymphoproliferative response directed against gelatin were performed at enrolment and on follow up.

Results None of the subjects had antibody to gelatin at enrolment and none developed gelatin IgE or IgG during follow‐up. There was no significant difference in the SIs of the subjects receiving gelatin DTaP (P = 0.150, 95% CI, −0.198–0.032), whereas lymphocyte activity to gelatin increased between enrolment and follow‐up in the subjects with non‐gelatin DTaP (P = 0.011, 95% CI, 0.063–0.338).

Conclusion Gelatin‐specific lymphocyte activity persists at comparable levels for more than 3 years in subjects who acquire a positive LPT response to gelatin after receiving primary DTaP vaccine containing gelatin. Furthermore, five out of eight subjects initially with negative LPT to gelatin have been shown to acquire specific LPT with time.
Island incidence and mainland population density: mammals from Mediterranean islands
Authors: Sara Maurizio , Morand Serge
Published: March 2003

Abstract.The reasons why some species are resistant to extinction or are better invaders of islands than others remain unexplained. In this study, we test the hypothesis that mammals living on the mainland at higher density than predicted by the density/body mass relationship have a much greater chance to colonize a small island successfully, and/or that they are less likely to become extinct when living on small islands. For this, we used data compiled on mammals from a number of Mediterranean islands. We show a nested pattern for mammals on western Mediterranean islands, which suggests that the distribution of mammals on these islands is not the result of a random process. Using two comparative methods, we show that mammal density on the mainland, corrected for body mass, is negatively correlated with island size. Mammals with a high density compared to the density/mass relationship are the best invaders and/or probably have less chance of going extinct on small islands when population size is small.
The price of autonomy
Authors: Baum Michael , Vaidya Jayant S.
Published: March 2003

The randomized controlled trial produces a clash of ethical principles with the need for informed consent (autonomy) in conflict with the principles of beneficence and justice. Informed consent is one of the major rate‐limiting factors of recruitment and this delays the discovery of life‐saving treatments indirectly. Whilst supporting the concept of non‐exploitation we wish to challenge the prevailing dogma by asking the awkward question ‘what is the price of autonomy?’. Using breast cancer as an example we have developed a decision model with explicit assumptions allowing numerical values to be fed into a mathematical equation, which calculates the cost in lives. With conservative assumptions we estimate that the price of autonomy is 2500 lives over a 10‐year period in the United Kingdom alone. We issue the challenge to health policy makers and ethicists to survey public opinion to determine the value placed on autonomy in the war against cancer.
Public involvement in health care priority setting: an overview of methods for eliciting values
Authors: Mullen Penelope M.
Published: March 2003

There is increasing interest, in the UK and elsewhere, in involving the public in health care priority setting. At the same time, however, there is evidence of lack of clarity about the objectives of some priority setting projects and also about the role of public involvement. Further, some projects display an apparent ignorance of both long‐standing theoretical literature and practical experience of methodologies for eliciting values in health care and related fields. After a brief examination of the context of health care priority setting and public involvement, this paper describes a range of different approaches to eliciting values. These approaches are critically examined on a number of dimensions including the type of choice allowed to respondents and the implications of aggregation of values across individuals. Factors which affect the appropriateness of the different techniques to specific applications are discussed. A check‐list of questions to be asked when selecting techniques is presented.
Giving tape recordings or written summaries of consultations to people with cancer: a systematic review
Authors: Scott J. Tim , Entwistle Vikki A. , Sowden Amanda J. , Watt Ian
Published: March 2003

Objective To examine the effects of providing recordings or summaries of consultations to people with cancer and their families.

Design Systematic review.

Data sources MEDLINE, CINAHL, Cancerlit, EMBASE and other electronic bibliographic databases. Bibliographies of relevant papers.

Selection criteria Randomized and non‐randomized controlled trials of the provision of taped recordings or written summaries of consultations to people with cancer and/or their families.

Main results Eight randomized controlled trials were found, all involving adult participants. No non‐randomized controlled trials were found. The quality of the studies was generally poor. Between 83% and 96% of people who received recordings or summaries found them useful to remind them of what was said and/or to inform family members and friends about their illness and treatment. Of seven studies that assessed recall of information given during the consultation, four reported better recall among the groups that received recordings or summaries than among control groups. Receiving a recording or summary had no significant effect on anxiety or depression between the groups. None of the included studies assessed survival or health outcomes other than psychological outcomes.

Conclusions Wider use of consultation tapes and summary letters could benefit many adults with cancer, without causing additional anxiety or depression, but consideration should be given to individuals’ circumstances and preferences.
Uncoupling of protein‐3 induces an uncontrolled uncoupling of mitochondria after expression in muscle derived L6 cells
Authors: Guerini Danilo , Prati Elisabetta , Desai Urvi , Nick Hans Peter , Flammer Rolf , Grüninger Stephan , Cumin Frederic , Kaleko Machael , Connelly Sheila , Chiesi Michele
Published: March 2003

The uncoupling proteins (UCPs) are thought to uncouple oxidative phosphorylation in the mitochondria and thus generate heat. One of the UCP isoforms, UCP3, is abundantly expressed in skeletal muscle, the major thermogenic tissue in humans. UCP3 has been overexpressed at high levels in yeast systems, where it leads to the uncoupling of cell respiration, suggesting that UCP3 may indeed be capable of dissipating the mitochondrial proton gradient. This effect, however, was recently shown to be a consequence of the high level of expression and incorrect folding of the protein and not to its intrinsic uncoupling activity. In the present study, we investigated the properties of UCP3 overexpressed in a relevant mammalian host system such as the rat myoblast L6␣cell line. UCP3 was expressed in relatively low levels (< 1 µg·mg−1 membrane protein) with the help of an adenovirus vector. Immunofluorescence microscopy of transduced L6 cells showed that UCP3 was expressed in more than 90% of the cells and that its staining pattern was characteristic for mitochondrial localization. The oxygen consumption of L6 cells under nonphosphorylating conditions increased concomitantly with the levels of UCP3 expression. However, uncoupling was associated with an inhibition of the maximal respiratory capacity of mitochondria and was not affected by purine nucleotides and free fatty acids. Moreover, recombinant UCP3 was resistant to Triton X‐100 extraction under conditions that fully solubilize membrane bound proteins.

 Thus, UCP3 can be uniformly overexpressed in the mitochondria of a relevant muscle‐derived cell line resulting in the expected increase of mitochondrial uncoupling. However, our data suggest that the protein is present in an incompetent conformation.
Ornithine decarboxylase‐antizyme is rapidly degraded through a mechanism that requires functional ubiquitin‐dependent proteolytic activity
Authors: Gandre Shilpa , Bercovich Zippi , Kahana Chaim
Published: March 2003

Antizyme is a polyamine‐induced cellular protein that binds to ornithine decarboxylase (ODC), and targets it to rapid ubiquitin‐independent degradation by the 26S proteasome. However, the metabolic fate of antizyme is not clear. We have tested the stability of antizyme in mammalian cells. In␣contrast with previous studies demonstrating stability in␣vitro in a reticulocyte lysate‐based degradation system, in cells antizyme is rapidly degraded and this degradation is inhibited by specific proteasome inhibitors. While the degradation of ODC is stimulated by the presence of cotransfected antizyme, degradation of antizyme seems to be independent of ODC, suggesting that antizyme degradation does not occur while presenting ODC to the 26S proteasome. Interestingly, both species of antizyme, which represent initiation at two in‐frame initiation codons, are rapidly degraded. The degradation of both antizyme proteins is inhibited in ts20 cells containing a thermosensitive ubiquitin‐activating enzyme, E1. Therefore we conclude that in contrast with ubiquitin‐independent degradation of ODC, degradation of antizyme requires a functional ubiquitin system.
Interaction of decorin with CNBr peptides from collagens I and II
Authors: Tenni Ruggero , Viola Manuela , Welser Franz , Sini Patrizia , Giudici Camilla , Rossi Antonio , Tira M. Enrica
Published: March 2003

Decorin is a small leucine‐rich chondroitin/dermatan sulfate proteoglycan reported to interact with fibrillar collagens through its protein core and to localize at d and e bands of the collagen fibril banding pattern. Using a solid‐phase assay, we have determined the interaction of peptides derived by CNBr cleavage of type I and type II collagen with decorin extracted from bovine tendon and its protein core and with a recombinant decorin preparation. At least five peptides have been found to interact with all three decorin samples. The interaction of peptides with tendon decorin has a dissociation constant in the nanomolar range. The triple helical conformation of the peptide trimeric species is a necessary requisite for the binding. All positive peptides have a region within the d and e bands of collagen fibrils. Two chemical derivatives of collagens and of positive peptides were prepared by N‐acetylation and N‐methylation of the primary amino group of Lys/Hyl side chains. Chemical modifications performed in mild conditions do not significantly alter the thermal stability of peptide trimeric species whereas they affect the interaction with decorin: N‐acetylation eliminates both the positive charge and the binding to decorin, whereas N‐methylation preserves the cationic character and modulates the binding. We conclude that decorin makes contacts with multiple sites in type I collagen and probably also in type II collagen and that some collagen Lys/Hyl residues are essential for the binding.
Variations in receptor site‐3 on rat brain and insect sodium channels highlighted by binding of a funnel‐web spider δ‐atracotoxin
Authors: Gilles Nicolas , Harrison Greg , Karbat Izhar , Gurevitz Michael , Nicholson Graham M. , Gordon Dalia
Published: March 2003

δ‐Atracotoxins (δ‐ACTXs) from Australian funnel‐web spiders differ structurally from scorpion α‐toxins (ScαTx) but similarly slow sodium current inactivation and compete for their binding to sodium channels at receptor site‐3. Characterization of the binding of 125I‐labelled δ‐ACTX‐Hv1a to various sodium channels reveals a decrease in affinity for depolarized (0 mV; Kd=6.5 ± 1.4 nm) vs.polarized (−55 mV; Kd=0.6 ± 0.2 nm) rat brain synaptosomes. The increased Kd under depolarized conditions correlates with a 4.3‐fold reduction in the association rate and a 1.8‐increase in the dissociation rate. In comparison, ScαTx binding affinity decreased 33‐fold under depolarized conditions due to a 48‐fold reduction in the association rate. The binding of 125I‐labelled δ‐ACTX‐Hv1a to rat brain synaptosomes is inhibited competitively by classical ScαTxs and allosterically by brevetoxin‐1, similar to ScαTx binding. However, in contrast with classical ScαTxs, 125I‐labelled δ‐ACTX‐Hv1a binds with high affinity to cockroach Na+ channels (Kd=0.42 ± 0.1 nm) and is displaced by the ScαTx, LqhαIT, a well‐defined ligand of insect sodium channel receptor site‐3. However, δ‐ACTX‐Hv1a exhibits a surprisingly low binding affinity to locust sodium channels. Thus, unlike ScαTxs, which are capable of differentiating between mammalian and insect sodium channels, δ‐ACTXs differentiate between various insect sodium channels but bind with similar high affinity to rat brain and cockroach channels. Structural comparison of δ‐ACTX‐Hv1a to ScαTxs suggests a similar putative bioactive surface but a ‘slimmer’ overall shape of the spider toxin. A slimmer shape may ease the interaction with the cockroach and mammalian receptor site‐3 and facilitate its association with different conformations of the rat brain receptor, correlated with closed/open and slow‐inactivated channel states.
Identification of novel membrane proteins by searching for patterns in hydropathy profiles
Authors: Clements John D. , Martin Rowena E.
Published: March 2003

A technique has been developed to search a proteome database for new members of a functional class of membrane protein. It takes advantage of the highly conserved secondary structure of functionally related membrane proteins. Such proteins typically have the same number of transmembrane domains located at similar relative positions in their polypeptide sequence. This gives rise to a characteristic pattern of peaks in their hydropathy profiles. To conduct a search, each member of a polypeptide database is converted to a hydropathy profile, peaks are automatically detected, and the pattern of peaks is compared with a template. A template was designed for the acetylcholine (ACh) and glycine receptors of the cys‐loop receptor superfamily. The key feature was a closely spaced triplet of hydropathy peaks bracketed by deep valleys. When applied to the human proteome the search procedure retrieved 153 profiles with a receptor‐like triplet of peaks. The approach was highly selective with 70% of the retrieved profiles annotated as known or putative receptors. These included ACh, glycine, γ‐amino butyric acid and seretonin receptors, which are all related by sequence. However, ionotropic glutamate receptors, which have almost no sequence homology with ACh receptors, were also retrieved. Thus, the strategy can find members of a functional class that cannot be identified by sequence alignment. To demonstrate that the strategy can easily be extended to other membrane protein families, a template was developed for the neurotransmitter/Na+ symporter family, and similar results were obtained. This approach should prove a useful adjunct to sequence‐based retrieval tools when searching for novel membrane proteins.
Effect of adenosine 5′‐[β,γ‐imido]triphosphate on myosin head domain movements
Authors: Hartvig Nóra , Lõrinczy Dénes , Farkas Nelli , Belagyi Joseph
Published: March 2003

Conventional and saturation transfer electron paramagnetic resonance spectroscopy (EPR and ST EPR) was used to study the orientation of probe molecules in muscle fibers in different intermediate states of the ATP hydrolysis cycle. A separate procedure was used to obtain ST EPR spectra with precise phase settings even in the case of samples with low spectral intensity.

Fibers prepared from rabbit psoas muscle were labeled with isothiocyanate spin labels at the reactive thiol sites of the catalytic domain of myosin. In comparison with rigor, a significant difference was detected in the orientation‐dependence of spin labels in the ADP and adenosine 5′‐[β,γ‐imido]triphosphate (AdoPP[CH2]P) states, indicating changes in the internal dynamics and domain orientation of myosin. In the AdoPP[CH2]P state, approximately half of the myosin heads reflected the motional state of ADP–myosin, and the other half showed a different dynamic state with greater mobility.
Activation of a covalent outer membrane phospholipase A dimer
Authors: Kingma Roelie L. , Egmond Maarten R.
Published: March 2003

The activity of outer membrane phospholipase A (OMPLA) is regulated by reversible dimerization. However, native OMPLA reconstituted in phospholipid vesicles was found to be present as a dimer but nevertheless inactive. To investigate the importance of dimerization for control of OMPLA activity, a covalent OMPLA dimer was constructed and its properties were compared to native OMPLA both in a micellar detergent and after reconstitution in a phospholipid bilayer. Unlike native OMPLA, activity of the covalent OMPLA dimer was independent of type and concentration of detergent in micellar systems. In such systems, the covalent OMPLA dimer invariantly displayed high calcium affinity. In contrast, high calcium concentrations were required to activate a covalent OMPLA dimer when present in intact vesicles. Solubilization of the vesicles increased the affinity for calcium, suggesting that in an intact bilayer the dimer interface is not properly formed. This was supported by the observation that OMPLA variants having an impaired dimeric interface also lacked high affinity calcium binding. A covalent linkage was not able to restore high affinity calcium binding in these variants, demonstrating that a proper dimer interface is essential for optimal catalysis.
The amyloid precursor protein interacts with neutral lipids
Authors: Lahdo Raghda , Coillet‐Matillon Stéphane , Chauvet Jean‐Paul , de La Fournière‐Bessueille Laurence
Published: March 2003

The amyloid protein precursor (APP) was incorporated into liposomes or phospholipid monolayers. APP insertion into liposomes required neutral lipids, such as l‐α‐phosphatidylcholine, in the target membrane. It was prevented in vesicles containing l‐α‐phosphatidylserine. The insertion was enhanced in acidic solutions, suggesting that it is modulated by specific charge/charge interactions. Surface‐active properties and behaviour of APP were characterized during insertion of the protein in monomolecular films of l‐α‐phosphatidylcholine, l‐α‐phosphatidylethanolamine or l‐α‐phosphatidylserine. The presence of the lipid film enhanced the rate of adsorption of the protein at the interface, and the increase in surface pressure was consistent with APP penetrating the lipid film. The adsorption of APP on the lipid monolayers displayed a significant head group dependency, suggesting that the changes in surface pressure produced by the protein were probably affected by electrostatic interactions with the lipid layers. Our results indicate that the penetration of the protein into the lipid monolayer is also influenced by the hydrophobic interactions between APP and the lipid. CD spectra showed that a large proportion of the α‐helical secondary structure of APP remained preserved over the pH or ionic strength ranges used. Our findings suggest that APP/membrane interactions are mediated by the lipid composition and depend on both electrostatic and hydrophobic effects, and that the variations observed are not due to major secondary structural changes in APP. These observations may be related to the partitioning of APP into membrane microdomains.
Progestin upregulates G‐protein‐coupled receptor 30 in breast cancer cells
Authors: Ahola Tytti M. , Purmonen Sami , Pennanen Pasi , Zhuang Ya‐Hua , Tuohimaa Pentti , Ylikomi Timo
Published: March 2003

A differential display method was used to study genes the expression of which is altered during growth inhibition induced by medroxyprogesterone acetate (MPA). A transcript of G‐protein‐coupled receptor 30 (GPR30) was upregulated by MPA in estrogen‐treated MCF‐7 breast cancer cells. Northern‐blot analysis showed a progestin‐specific primary target gene, which was enhanced by progesterone and different progestins, but not by dihydrotestosterone or dexamethasone, and which was abrogated by antiprogestin RU486. The dose‐dependent and time‐dependent increase in GPR30 mRNA expression correlated with MPA‐induced growth inhibition in MCF‐7 cells. Additionally, GPR30 upregulation by progestin correlated with growth inhibition when a comparison was made between different breast cancer cell lines. The ERK1/ERK2 pathway is capable of inducing progesterone receptor‐dependent and ligand‐dependent transcription. Thus we sought to establish whether different MAPK pathway inhibitors affect progestin‐induced GPR30 mRNA regulation. The regulation of GPR30 was independent of ERK pathway activation, but the p38 pathway inhibitor induced GPR30 expression, which suggested a potential gene regulation pathway. These data demonstrate a new progestin target gene, the expression of which correlates with growth inhibition.
Caged O2
Authors: Ludovici Claudia , Fröhlich Roland , Vogtt Karsten , Mamat Björn , Lübben Mathias
Published: March 2003

We developed the synthesis of the caged oxygen donor (µ‐peroxo)(µ‐hydroxo)bis[bis(bipyridyl)cobalt(III)] complex (HPBC) as nitrate salt, which has, compared with the perchlorate‐form described previously [MacArthur, R., Sucheta, A., Chong, F.F. & Einarsdottir, Ö. (1995) Proc. Natl Acad. Sci. USA, 92, 8105–8109], greatly enhanced solubility. Now, the quantum efficiency of the photolytical release of dioxygen was determined to be 0.4 per photon at a laser wavelength of 308 nm, which was used to observe biological reactions. The X‐ray structure of HPBC has been solved, and the molecular interactions of photochemically generated oxygen with cytochrome oxidase were investigated with optical and FT‐IR spectroscopy: it acts as acceptor of electrons transferred from prereduced cytochrome bo3, the heme‐copper oxidase from Escherichia coli. FT‐IR spectra revealed typical absorbance difference changes in the carbonyl region of cytochrome bo3, supported by bandshifts due to solvent isotope exchange and by assignment using site‐directed mutants. IR difference spectra of the photooxidation reaction using the caged oxygen compound, and of the photoreduction reaction using the caged electron donor FMN, have inverted shapes. The spectroscopic signals of carboxyl groups are thus equivalent in both reactions: the use of chemically produced oxygen allows the observation of the ongoing molecular changes of cytochrome bo3 oxidase under quasi‐physiological conditions.
In vitro gene therapy of mucopolysaccharidosis type I by lentiviral vectors
Authors: Di Natale Paola , Di Domenico Carmela , Villani Guglielmo R. D. , Lombardo Angelo , Follenzi Antonia , Naldini Luigi
Published: March 2003

Mucopolysaccharidosis type I (MPS I) results from a deficiency in the enzyme α‐l‐iduronidase (IDUA), and is characterized by skeletal abnormalities, hepatosplenomegaly and neurological dysfunction. In this study, we used a late generation lentiviral vector to evaluate the utility of this vector system for the transfer and expression of the human IDUA cDNA in MPS I fibroblasts. We observed that the level of enzyme expression in transduced cells was 1.5‐fold the level found in normal cells; the expression persisted for at least two months. In addition, transduced MPS I fibroblasts were capable of clearing intracellular radiolabeled glycosaminoglycan (GAG). Pulse‐chase experiments on transduced fibroblasts showed that the recombinant enzyme was synthesized as a 76‐kDa precursor form and processed to a 66‐kDa mature form; it was released from transduced cells and was endocytosed into a second population of untreated MPS I fibroblasts via a mannose 6‐phosphate receptor. These results suggest that the lentiviral vector may be used for the delivery and expression of the IDUA gene to cells in vivo for treatment of MPS I.
Interaction of bovine coagulation factor X and its glutamic‐acid‐containing fragments with phospholipid membranes
Authors: Erb Eva‐Maria , Stenflo Johan , Drakenberg Torbjörn
Published: March 2003

The interaction of blood coagulation factor X and its Gla‐containing fragments with negatively charged phospholipid membranes composed of 25 mol% phosphatidylserine (PtdSer) and 75 mol% phosphatidylcholine (PtdCho) was studied by surface plasmon resonance. The binding to 100 mol% PtdCho membranes was negligible. The calcium dependence in the membrane binding was evaluated for intact bovine factor X (factor X) and the fragment containing the Gla‐domain and the N‐terminal EGF (epidermal growth factor)‐like domain, Gla–EGFN, from factor X. Both proteins show the same calcium dependence in the membrane binding. Calcium binding is cooperative and half‐maximum binding was observed at 1.5 mm and 1.4 mm, with the best fit to the experimental data with three cooperatively bound calcium ions for both the intact protein and the fragment. The dissociation constant (Kd) for binding to membranes containing 25 mol% PtdSer decreased from 4.6 µm for the isolated Gla‐domain to 1 µm for the fragments Gla–EGFN and Gla–EGFNC (the Gla‐domain and both EGF‐like domains) fragments and to 40 nm for the entire protein as zymogen, activated enzyme or in the active‐site inhibited form. Analysis of the kinetics of adsorption and desorption confirmed the equilibrium binding data.
Determinants of the inhibition of a Taiwan habu venom metalloproteinase by its endogenous inhibitors revealed by X‐ray crystallography and synthetic inhibitor analogues
Authors: Huang Kai‐Fa , Chiou Shyh‐Horng , Ko Tzu‐Ping , Wang Andrew H.‐J.
Published: March 2003

Venoms from crotalid and viperid snakes contain several peptide inhibitors which regulate the proteolytic activities of their snake‐venom metalloproteinases (SVMPs) in a reversible manner under physiological conditions. In this report, we describe the high‐resolution crystal structures of a SVMP, TM‐3, from Taiwan habu (Trimeresurus mucrosquamatus) cocrystallized with the endogenous inhibitors pyroGlu‐Asn‐Trp (pENW), pyroGlu‐Gln‐Trp (pEQW) or pyroGlu‐Lys‐Trp (pEKW). The binding of inhibitors causes some of the residues around the inhibitor‐binding environment of TM‐3 to slightly move away from the active‐site center, and displaces two metal‐coordinated water molecules by the C‐terminal carboxylic group of the inhibitors. This binding adopts a retro‐manner principally stabilized by four possible hydrogen bonds. The Trp indole ring of the inhibitors is stacked against the imidazole of His143 in the S−1 site of the proteinase. Results from the study of synthetic inhibitor analogues showed the primary specificity of Trp residue of the inhibitors at the P−1 site, corroborating the stacking effect observed in our structures. Furthermore, we have made a detailed comparison of our structures with the binding modes of other inhibitors including batimastat, a hydroxamate inhibitor, and a barbiturate derivative. It suggests a close correlation between the inhibitory activity of an inhibitor and its ability to fill the S−1 pocket of the proteinase. Our work may provide insights into the rational design of small molecules that bind to this class of zinc‐metalloproteinases.
IgE reactivity of tandem repeats derived from cockroach allergen, Bla g 1
Authors: Pomés Anna , Vailes Lisa D. , Helm Ricki M. , Chapman Martin D.
Published: March 2003

Sensitization to cockroach allergens is associated with the development of asthma. Bla g 1 is a German cockroach allergen that shows allergenic cross‐reactivity with American cockroach allergen, Per a 1, and has a molecular structure composed of multiple tandem amino‐acid repeats. Two consecutive repeats are not identical but form a duplex that constitutes a basic molecular unit of Bla g 1. By molecular mass, purified natural Bla g 1 would contain approximately two duplexes. We investigated the pattern of IgE antibody binding to this repeated structure, and whether one or two duplexes are sufficient for IgE binding. Recombinant (r)Bla g 1 duplexes were expressed in Escherichia coli and in Pichia pastoris, and analyzed for monoclonal antibody and IgE antibody binding by ELISA and/or immunoblotting. Optimal rBla g 1 expression was obtained using methanol‐inducible P. pastoris (> 95% pure protein, yield ≈ 48 mg·L−1), and rBla g 1 was produced as multiple molecular forms of molecular mass 43, 32, 21 and 6 kDa, that were the result of proteolytic cleavage. There was an excellent correlation between IgE antibody binding to natural and recombinant Bla g 1 (r = 0.91, n = 29, P < 0.001), and immunoblot analysis showed that a single Bla g 1 duplex was sufficient for IgE antibody binding. The rBla g 1 is suitable for structural studies and a candidate for clinical use in diagnosis of cockroach allergy and development of new forms of immunotherapy.
Transcriptional regulation of erythropoiesis
Authors: Perry Chava , Soreq Hermona
Published: March 2003

Haematopoiesis, the differentiation of haematopoietic stem cells and progenitors into various lineages, involves complex interactions of transcription factors that modulate the expression of downstream genes and mediate proliferation and differentiation signals. Commitment of pluripotent haematopoietic stem cells to the erythroid lineage induces erythropoiesis, the production of red blood cells. This process involves a concerted progression through an erythroid burst forming unit (BFU‐E), an erythroid colony forming unit (CFU‐E), proerythroblast and an erythroblast. The terminally differentiated erythrocytes, in mammals, lose their nucleus yet function several more months. A well‐coordinated cohort of transcription factors regulates the formation, survival, proliferation and differentiation of multipotent progenitor into the erythroid lineage. Here, we discuss broad‐spectrum factors essential for self‐renewal and/or differentiation of multipotent cells as well as specific factors required for proper erythroid development. These factors may operate solely or as part of transcriptional complexes, and exert activation or repression. Sequence comparisons reveal evolutionarily conserved modular composition for these factors; X‐ray crystallography demonstrates that they include multidomain elements (e.g. HLH or zinc finger motifs), consistent with their complex interactions with other proteins. Finally, transfections and genomic studies show that the timing of each factor's expression during the hematopoietic process, the cell lineages affected and the existing combination of other factors determine the erythroid cell fate.
Pyruvate decarboxylase from Kluyveromyces lactis
Authors: Krieger Florian , Spinka Michael , Golbik Ralph , Hübner Gerhard , König Stephan
Published: March 2003

Pyruvate decarboxylase (EC 4.1.1.1) was isolated and purified from the yeast Kluyveromyces lactis. The properties of this enzyme relating to the native oligomeric state, the subunit size, the nucleotide sequence of the coding gene(s), the catalytic activity, and protein fluorescence as well as circular dichroism are very similar to those of the well characterized pyruvate decarboxylase species from yeast. Remarkable differences were found in the substrate activation behaviour of the two pyruvate decarboxylases using three independent methods: steady‐state kinetics, stopped‐flow measurements, and kinetic dilution experiments. The dependence of the observed activation rate constant on the substrate concentration of pyruvate decarboxylase from K. lactis showed a minimum at a pyruvate concentration of 1.5 mm. According to the mechanism of substrate activation suggested this local minimum occurs due to the big ratio of the dissociation constants for the binding of the first (regulatory) and the second (catalytic) substrate molecule. The microscopic rate constants of the substrate activation could be determined by a refined fit procedure. The influence of the artificial activator pyruvamide on the activation of the enzyme was studied.
Changes in ultrastructure and the occurrence of permeability transition in mitochondria during rat liver regeneration
Authors: Guerrieri Ferruccio , Pellecchia Giovanna , Lopriore Barbara , Papa Sergio , Esterina Liquori Giuseppa , Ferri Domenico , Moro Loredana , Marra Ersilia , Margherita Greco
Published: March 2003

Mitochondrial bioenergetic impairment has been found in the organelles isolated from rat liver during the prereplicative phase of liver regeneration. To gain insight into the mechanism underlying this impairment, we investigated mitochondrial ultrastructure and membrane permeability properties in the course of liver regeneration after partial hepatectomy, with special interest to the role played by Ca2+ in this process. The results show that during the first day after partial hepatectomy, significant changes in the ultrastructure of mitochondria in situ occur. Mitochondrial swelling and release from mitochondria of both glutamate dehydrogenase and aspartate aminotransferase isoenzymes with an increase in the mitochondrial Ca2+ content were also observed. Cyclosporin‐A proved to be able to prevent the changes in mitochondrial membrane permeability properties. At 24 h after partial hepatectomy, despite alteration in mitochondrial membrane permeability properties, no release of cytochrome c was found. The ultrastructure of mitochondria, the membrane permeability properties and the Ca2+ content returned to normal values during the replicative phase of liver regeneration. These results suggest that, during the prereplicative phase of liver regeneration, the changes in mitochondrial ultrastructure observed in liver specimens were correlated with Ca2+‐induced permeability transition in mitochondria.
Recombinant human glucose‐6‐phosphate dehydrogenase
Authors: Wang Xiao‐Tao , Au Shannon W. N. , Lam Veronica M. S. , Engel Paul C.
Published: March 2003

Cloning and over‐expression of human glucose 6‐phosphate dehydrogenase (Glc6P dehydrogenase) has for the first time allowed a detailed kinetic study of a preparation that is genetically homogeneous and in which all the protein molecules are of identical age. The steady‐state kinetics of the recombinant enzyme, studied by fluorimetric initial‐rate measurements, gave converging linear Lineweaver–Burk plots as expected for a ternary‐complex mechanism. Patterns of product and dead‐end inhibition indicated that the enzyme can bind NADP+ and Glc6P separately to form binary complexes, suggesting a random‐order mechanism. The Kd value for the binding of NADP+ measured by titration of protein fluorescence is 8.0 µm, close to the value of 6.8 µm calculated from the kinetic data on the assumption of a rapid‐equilibrium random‐order mechanism. Strong evidence for this mechanism and against either of the compulsory‐order possibilities is provided by repeating the kinetic analysis with each of the natural substrates replaced in turn by structural analogues. A full kinetic analysis was carried out with deaminoNADP+ and with deoxyglucose 6‐phosphate as the alternative substrates. In each case the calculated dissociation constant upon switching a substrate in a random‐order mechanism (e.g. that for NADP+ upon changing the sugar phosphate) was indeed constant within experimental error as expected. The calculated rate constants for binding of the leading substrate in a compulsory‐order mechanism, however, did not remain constant when the putative second substrate was changed. Previous workers, using enzyme from pooled blood, have variously proposed either compulsory‐order or random‐order mechanisms. Our study appears to provide unambiguous evidence for the latter pattern of substrate binding.
Substrate selectivity and sensitivity to inhibition by FK506 and cyclosporin A of calcineurin heterodimers composed of the α or β catalytic subunit
Authors: Perrino Brian A. , Wilson Andrew J. , Ellison Patricia , Clapp Lucie H.
Published: March 2003

The calcineurin (CaN) α and β catalytic subunit isoforms are coexpressed within almost all cell types. The enzymatic properties of CaN heterodimers comprised of the regulatory B subunit (CnB) with either the α or β catalytic subunit were compared using in vitro phosphatase assays. CaN containing the α isoform (CnAα) has lower Km and higher Vmax values than CaN containing the β isoform (CnAβ) toward the PO4‐RII, PO4‐DARPP‐32(20–38) peptides, and p‐nitrophenylphosphate (pNPP). CaN heterodimers containing the α or β catalytic subunit isoform displayed identical calmodulin dissociation rates. Similar inhibition curves for each CaN heterodimer were obtained with the CaN autoinhibitory peptide (CaP) and cyclophilin A/cyclosporin A (CyPA/CsA) using each peptide substrate at Km concentrations, except for a five‐ to ninefold higher IC50 value measured for CaN containing the β isoform with p‐nitrophenylphosphate as substrate. No difference in stimulation of phosphatase activity toward p‐nitrophenylphosphate by FKBP12/FK506 was observed. At low concentrations of FKBP12/FK506, CaN containing the α isoform is more sensitive to inhibition than CaN containing the β isoform using the phosphopeptide substrates. Higher concentrations of FKBP12/FK506 are required for maximal inhibition of β CaN using PO4‐DARPP‐32(20–38) as substrate. The functional differences conferred upon CaN by the α or β catalytic subunit isoforms suggest that the α:β and CaN:substrate ratios may determine the levels of CaN phosphatase activity toward specific substrates within tissues and specific cell types. These findings also indicate that the α and β catalytic subunit isoforms give rise to substrate‐dependent differences in sensitivity toward FKBP12/FK506.
Cytochrome c from a thermophilic bacterium has provided insights into the mechanisms of protein maturation, folding, and stability
Authors: Sambongi Yoshihiro , Uchiyama Susumu , Kobayashi Yuji , Igarashi Yasuo , Hasegawa Jun
Published: March 2003

Cytochrome c is widely distributed in bacterial species, from mesophiles to thermophiles, and is one of the best‐characterized redox proteins in terms of biogenesis, folding, structure, function, and evolution. Experimental molecular biology techniques (gene cloning and expression) have become applicable to cytochrome c, enabling its engineering and manipulation. Heterologous expression systems for cytochromes c in bacteria, for use in mutagenesis studies, have been established by extensive investigation of the biological process by which the functional structure is formed. Mutagenesis and structure analyses based on comparative studies using a thermophile Hydrogenobacter thermophilus cytochrome c‐552 and its mesophilic counterpart have provided substantial clues to the mechanism underlying protein stability at the amino‐acid level. The molecular mechanisms underlying protein maturation, folding, and stability in bacterial cytochromes c are beginning to be understood.
A critical motif for oligomerization and chaperone activity of bacterial α‐heat shock proteins
Authors: Studer Sonja , Obrist Markus , Lentze Nicolas , Narberhaus Franz
Published: March 2003

Oligomerization into multimeric complexes is a prerequisite for the chaperone function of almost all α‐crystallin type heat shock proteins (α‐Hsp), but the molecular details of complex assembly are poorly understood. The α‐Hsp proteins from Bradyrhizobium japonicum are suitable bacterial models for structure‐function studies of these ubiquitous stress proteins. They fall into two distinct classes, A and B, display chaperone activity in vitro and form oligomers of ≈ 24 subunits. We constructed 19 derivatives containing truncations or point mutations within the N‐ and C‐terminal regions and analyzed them by gel filtration, citrate synthase assay and coaffinity purification. Truncation of more than the initial few amino acids of the N‐terminal region led to the formation of distinct dimeric to octameric structures devoid of chaperone activity. In the C‐terminal extension, integrity of an isoleucine‐X‐isoleucine (I‐X‐I) motif was imperative for α‐Hsp functionality. This I‐X‐I motif is one of the characteristic consensus motifs of the α‐Hsp family, and here we provide experimental evidence of its structural and functional importance. α‐Hsp proteins lacking the C‐terminal extension were inactive, but still able to form dimers. Here, we demonstrate that the central α‐crystallin domain alone is not sufficient for dimerization. Additional residues at the end of the N‐terminal region were required for the assembly of two subunits.
The solution structure and activation of visual arrestin studied by small‐angle X‐ray scattering
Authors: Shilton Brian H. , McDowell J. Hugh , Smith W. Clay , Hargrave Paul A.
Published: March 2003

Visual arrestin is converted from a ‘basal’ state to an ‘activated’ state by interaction with the phosphorylated C‐terminus of photoactivated rhodopsin (R*), but the conformational changes in arrestin that lead to activation are unknown. Small‐angle X‐ray scattering (SAXS) was used to investigate the solution structure of arrestin and characterize changes attendant upon activation. Wild‐type arrestin forms dimers with a dissociation constant of 60 µm. Small conformational changes, consistent with local movements of loops or the mobile N‐ or C‐termini of arrestin, were observed in the presence of a phosphopeptide corresponding to the C‐terminus of rhodopsin, and with an R175Q mutant. Because both the phosphopeptide and the R175Q mutation promote binding to unphosphorylated R*, we conclude that arrestin is activated by subtle conformational changes. Most of the arrestin will be in a dimeric state in vivo. Using the arrestin structure as a guide [Hirsch, J.A., Schubert, C., Gurevich, V.V. & Sigler, P.B. (1999) Cell
97, 257–269], we have identified a model for the arrestin dimer that is consistent with our SAXS data. In this model, dimerization is mediated by the C‐terminal domain of arrestin, leaving the N‐terminal domains free for interaction with phosphorylated R*.
A cocaine insensitive chimeric insect serotonin transporter reveals domains critical for cocaine interaction
Authors: Sandhu Sumandeep K. , Ross Linda S. , Gill Sarjeet S.
Published: March 2003

Serotonin transporters are key target sites for clinical drugs and psychostimulants, such as fluoxetine and cocaine. Molecular cloning of a serotonin transporter from the central nervous system of the insect Manduca sexta enabled us to define domains that affect antagonist action, particularly cocaine. This insect serotonin transporter transiently expressed in CV‐1 monkey kidney cells exhibits saturable, high affinity Na+ and Cl– dependent serotonin uptake, with estimated Km and Vmax values of 436 ± 19 nm and 3.8 ± 0.6 × 10−18 mol·cell·min−1, respectively. The Manduca high affinity Na+/Cl– dependent transporter shares 53% and 74% amino acid identity with the human and fruit fly serotonin transporters, respectively. However, in contrast to serotonin transporters from these two latter species, the Manduca transporter is inhibited poorly by fluoxetine (IC50 = 1.23 µm) and cocaine (IC50 = 12.89 µm). To delineate domains and residues that could play a role in cocaine interaction, the human serotonin transporter was mutated to incorporate unique amino acid substitutions, detected in the Manduca homologue. We identified a domain in extracellular loop 2 (amino acids 148–152), which, when inserted into the human transporter, results in decreased cocaine sensitivity of the latter (IC50 = 1.54 µm). We also constructed a number of chimeras between the human and Manduca serotonin transporters (hSERT and MasSERT, respectively). The chimera, hSERT1–146/MasSERT106–587, which involved N‐terminal swaps including transmembrane domains (TMDs) 1 and 2, was remarkably insensitive to cocaine (IC50 = 180 µm) compared to the human (IC50 = 0.431 µm) and Manduca serotonin transporters. The chimera MasSERT1–67/hSERT109–630, which involved only the TMD1 swap, showed greater sensitivity to cocaine (IC50 = 0.225 µm) than the human transporter. Both chimeras showed twofold higher serotonin transport affinity compared to human and Manduca serotonin transporters. Our results show TMD1 and TMD2 affect the apparent substrate transport and antagonist sensitivity by possibly providing unique conformations to the transporter. The availability of these chimeras facilitates elucidation of specific amino acids involved in interactions with cocaine.
NADH oxidation and NAD+ reduction catalysed by tightly coupled inside‐out vesicles from Paracoccus denitrificans
Authors: Kotlyar Alexander B. , Borovok Natalia
Published: March 2003

Tightly coupled inside‐out vesicles were prepared from Paracoccus denitrificans cells (SPP, sub‐Paracoccus particles) and characterized kinetically. The rate of NADH oxidation, catalysed by SPP, increases 6–8 times on addition of gramicidin. The vesicles are capable of catalysing ΔµH+‐dependent reverse electron transfer from quinol to NAD+. The kinetic parameters of the NADH‐oxidase and the reverse electron transfer carried out by membrane‐bound P. denitrificans complex I were estimated and compared with those of the mitochondrial enzyme. The data demonstrate that catalytic properties of the dinucleotide‐binding site of the bacterial and mitochondrial complex I are almost identical, pointing out similar organization of the site in mammals and P. denitrificans. Inhibition of the bacterial complex I by a specific inhibitor of Q reduction, rotenone, is very different from that of the mitochondrial enzyme. The inhibitor is capable of suppressing the NADH oxidation reaction only at micromolar concentrations, while the activity of mitochondrial enzyme is suppressed by nanomolar concentrations of rotenone. In contrast to the mitochondrial enzyme, rotenone, even at concentrations as high as 10 µm, does not inhibit the reverse, ΔµH+‐dependent NAD+‐reductase reaction on SPP.
Domains of ERRγ that mediate homodimerization and interaction with factors stimulating DNA binding
Authors: Hentschke Moritz , Süsens Ute , Borgmeyer Uwe
Published: March 2003

The estrogen receptor‐related receptor γ (ERRγ/ERR3/NR3B3) is an orphan member of the nuclear receptor superfamily closely related to the estrogen receptors. To explore the DNA binding characteristics, the protein–DNA interaction was studied in electrophoretic mobility shift assays (EMSAs). In vitro translated ERRγ binds as a homodimer to direct repeats (DR) without spacing of the nuclear receptor half‐site 5′‐AGGTCA‐3′ (DR‐0), to extended half‐sites, and to the inverted estrogen response element. Using ERRγ deletion constructs, binding was found to be dependent on the presence of sequences in the ligand binding domain (LBD). A far‐Western analysis revealed that ERRγ forms dimers even in the absence of DNA. Two elements, located in the hinge region and in the LBD, respectively, are necessary for DNA‐independent dimerization. DNA binding of bacterial expressed ERRγ requires additional factors present in the serum and in cellular extracts. Fusion proteins of the germ cell nuclear factor (GCNF/NR6A1) with ERRγ showed that the characteristic feature to be stimulated by additional factors can be transferred to a heterologous protein. The stimulating activity was further characterized and its target sequence narrowed down to a small element in the hinge region.