bluuebunny/crossref_metadata_2025 huggingface dataset:
Atopic allergy and delayed type hypersensitivity in Estonian children
Authors: Julge K. , Meriste* S. , Kemp A. , Björkstén B.
Published: March 2003

Background A shift in the balance ofT helper (Th) cell subsets towards a polarized Th2 population is generally accepted to occur in atopic disease, however, both Th1 and Th2 disorders have increased over the past decades in Western communities.

Objective The aim of our study was to investigate delayed type hypersensitivity (DTH) response in atopic and non‐atopic children in a population with a low prevalence of allergic disorders.

Methods Skin prick tests (SPT) were performed with fresh egg white and extracts of five inhalant allergens, i.e. cat, dog, house dust mite (Dermatophagoides pteronyssinus), birch and timothy, and DTH response was evaluated by Multitest® CMI in 72 Estonian 4‐ to 6‐year‐old children.

Results The frequency of response to diphtheria was significantly increased in SPT‐positive children (55% vs. 26%, χ2 = 5.5; P = 0.038). The induration to diphtheria (2.4 ± 0.5 vs. 0.9 ± 0.2 mm; P = 0.004), and tetanus (3.5 ± 0.6 vs. 2.1 ± 0.3 mm; P = 0.025) was significantly greater in the SPT‐positive children. The cumulative size of induration in the positive DTH tests was significantly greater in the SPT‐positive children (9.0 ± 1.2 vs. 5.2 ± 0.6 mm, P = 0.01).

Conclusion In this group of children our findings do not support the hypothesis of an immune deviation with decreased Th1 and increased Th2 responses leading to atopic disease, but rather a process of immune modulation whereby both Th1 and Th2 responses are increased in atopic subjects.
Severe respiratory syncytial virus infection in early life is associated with increased type 2 cytokine production in Gambian children
Authors: Van Der Sande M. A. B. , Kidd I. M. , Goetghebuer T. , Martynoga R. A. , Magnusen A. , Allen S. , Weber M. W. , Fielding K. L. , Marchant A. , Whittle H. C.
Published: March 2003

Background Severe respiratory syncytial virus (RSV) infection in early childhood has been associated with subsequent wheezing and atopy. The aim of this study was to test if severe RSV infection in early life was associated with an increase in type 2 cytokine production and atopy in Gambian children 5 years later.

Methods A cohort of children with severe RSV infection during the first year of life (‘cases’, n = 66) and without (‘controls’, n = 122) was followed‐up at 5 years of age. Immediate hypersensitivity to common allergens, airway reactivity, serum IgE concentration and the production of IFN‐γ, IL‐5 and IL‐13 by lymphocytes activated in vitro with RSV F‐G or control antigens was determined.

Results After adjustment for confounders, cases produced significantly higher concentrations of IL‐13 in response to RSV F‐G and of IL‐5 and IL‐13 in response to tuberculin. Cases were more likely to have presented with a wheezy lower respiratory tract infection in the first 3 years of life (adjusted odds ratio = 9.9; 95% CI 1.6–61.0), but not thereafter. Cases and controls had similar skin response to allergens, airway reactivity and serum IgE concentrations.

Conclusion Severe RSV infection in early life is associated with a higher production of type 2 cytokines in Gambian children at 5 years of age. However this does not appear to result in increased risk of atopy or clinical allergy at that age.
Oral lichen planus: different clinical features in HCV‐positive and HCV‐negative patients
Authors: Mignogna Michele D. , Lo Muzio Lorenzo , Lo Russo Lucio ,   DDS, Fedele Stefano ,   DDS, Ruoppo Elvira ,   DDS, Bucci Eduardo
Published: March 2003

Background Hepatitis C virus (HCV) infection induces variable dermatologic manifestations.

Objective To determine whether differences exist in the clinical features and behavior of oral lichen planus (OLP) between HCV‐positive (HCV+ve) and HCV‐negative (HCV–ve) patients.

Methods Two hundred and sixty three patients (156 women and 107 men), with a mean age of 55.5 years, with OLP (76 HCV+ve and 187 HCV–ve) were clinically evaluated. Previously, all local factors that could modify the clinical characteristics were removed and were monitored carefully following morphology.

Results In both groups, the prevalent clinical form of OLP was the mixed form (33.1% in HCV–ve and 35.5% in HCV+ve patients), in which reticular–plaque lesions coexist with atrophic–erosive ones. The reticular form was more frequent in HCV+ve (25%) than in HCV–ve (18.7%) patients, whereas plaque lesions were more prevalent in HCV–ve (15.5%) than in HCV+ve (5.2%) patients (P < 0.01, chi‐squared test). There were no significant differences in the frequency of erosive (27.2% in HCV–ve and 27.6% in HCV+ve) and atrophic (5.3% in HCV–ve and 5.2% in HCV+ve) forms between the two groups.

Conclusions Our findings show that there were statistically significant differences between OLP‐HCV–ve and OLP‐HCV+ve groups for reticular and plaque clinical forms. These findings underline the importance of liver examination in all OLP patients, including cases with mild, asymptomatic keratotic forms of the disease.
Homology, biogeography and areas of endemism
Authors: Morrone Juan J.
Published: March 2003

Hypotheses of biogeographic homology constitute the basis of historical biogeography. Primary biogeographic homology refers to a conjecture on a common biogeographic history, and secondary biogeographic homology refers to the cladistic test of the formerly recognized homology. Panbiogeography deals with the former, through the recognition of generalized tracks and areas of endemism, whereas cladistic biogeography deals with the latter, through the generation of general area cladograms. A historical biogeographic analysis may include both approaches, in a two‐stage analysis.
A randomized community intervention trial to increase awareness and knowledge of the role of periconceptional folate in women of child‐bearing age
Authors: Watson Maxwell J. , Watson Lyndsey F. , Bell Robin J. , Halliday Jane L. , Burford Necia , Brennecke Shaun P.
Published: March 2003

Objectives To determine the effect of a consumer‐directed information campaign to increase knowledge of folate for the prevention of neural tube defects among women of child‐bearing age, and to measure women’s recall of sources of information and knowledge about folate.

Design A community randomized trial.

Setting Three matched pairs of geographically distinct Local Government Areas in the state of Victoria, Australia.

Intervention Printed information recommending folate intake to decrease the risk of neural tube defects was disseminated to women of child‐bearing age in three of the Local Government Areas selected randomly.

Main outcome measure The proportion of women aware of the association between folate and spina bifida.

Results Of 1197 women interviewed prior to the intervention, 12.4% (adjusted for the cluster and population sampling unit) were aware of folate and neural tube defects. After the intervention, there was not only a significant background increase of 3.4% (P=0.02) in folate awareness since the pre‐intervention survey (n=603), but also a significant additional increase of 4.0% (P=0.04) owing to the intervention itself (n=603). Only 70% of women who were aware of folate knew the correct timing.

Conclusions The provision of printed educational material can increase folate awareness among women of child‐bearing age. A comprehensive, long‐term and ongoing health promotion campaign including such material, together with initiatives by relevant health service providers and the food industry, could best address the current low levels of folate awareness among women of child‐bearing age.
Developing a prioritisation framework: experiences from a Scottish Health Authority
Authors: Scott Sheila N. , Lees Ann
Published: March 2003

Objectives To describe the development of the Prioritisation Scoring Index (PSI) and its use in a prioritisation framework, providing examples where it has been used to prioritise between bids from different specialities and to assist in decision‐making regarding funding of service developments. To outline lessons learned for other health authorities when developing their own prioritisation methodologies.

Background The PSI was designed for prioritising: investments and dis‐investments; non‐recurring and recurring monies as well as differing specialities, care groups and types of intervention.

Methods The PSI consists of a ‘basket’ of utility criteria and takes account of the numbers of people that would receive the proposed intervention and the marginal cost for each additional person receiving the intervention. A multidisciplinary panel scored and ranked the bids. Two rankings were produced for each intervention according to (1) the average panel score for the utility criteria and (2) the cost per additional person receiving the intervention. An average of these two rankings produced the overall PSI rankings.

Results Almost 200 bids, with a total value of £50 million, were ranked, using the PSI, to prioritise developments worth approximately £17.5 million that could be funded in a phased implementation through the Health Improvement Programme.

Conclusions Use of the PSI has allowed explicit prioritisation of development bids in substantial exercises for both non‐recurring and recurring funding. We describe steps to be considered when other health authorities are developing their own prioritisation frameworks.
Consumers’ views of quality in the consultation and their relevance to ‘shared decision‐making’ approaches
Authors: Edwards Adrian , Elwyn Glyn , Smith Christine , Williams Simon , Thornton Hazel
Published: March 2003

Background There is a recognized need to assess the effects of shared decision‐making and other communication interventions. However, the outcomes usually assessed for evidence of ‘effectiveness’ are determined by researchers and have not been based on consumers’ views.

Aim This study aimed to identify the important outcomes of consultations for consumers, and to compare with those reported in the current literature.

Setting and participants Forty‐seven participants attending six focus group interviews. Most interviews took place in and all were orientated towards the UK primary care setting.

Methods Focus group study.

Results Many affective outcomes were identified, consistent with the current literature trends. However, many cognitive and behavioural outcomes that are assessed in the current literature were not noted by participants as important. Furthermore, a broader range of outcomes than is evident in the current literature was viewed as important to these participants.

Conclusions There is a need to revisit the outcomes which are assessed in decision‐making and communication research. The outcomes of greatest importance to consumers must be identified and confirmed by new research which is based directly on the views of consumers themselves.
Mammography messages in popular media: implications for patient expectations and shared clinical decision‐making
Authors: Dobias Karen S. , Moyer Cheryl A. , McAchran Sarah E. , Katz Steven J. , Sonnad Seema S.
Published: March 2003

Objective To examine the relationship between the quantity and content of information about mammography in popular magazines and the educational level of their target audience.

Design Articles published in popular magazines from January 1988 through April 1994 in which ≥ 25% of all readers were females ≥ 35 years of age were identified (n=65). We used the proportion of readers who were college graduates to stratify the magazines into three education levels. We used a content analysis to assess the relationship between media messages about mammography and readers’ education levels.

Results Seventy‐eight percent of lowest education level articles were categorized as persuasive or prescriptive compared with 28% of articles in the highest education level (P < 0.01). Only 26% of the lowest education level articles that discussed screening guidelines for women under 50 years of age considered the issue controversial, while 59% of the high education level articles considered it controversial (P < 0.01).

Conclusion Women with lower education levels received a clearly persuasive or prescriptive message urging mammography screening, while higher educated women received more balanced and informative messages. Such differences suggest that women may be entering their physicians’ offices with very different sets of information from which to draw when faced with clinical decisions. Physicians and other health‐care providers should be aware of these potential differences, and further research should be done to explore the relationship between women’s preferences for participation in shared decision‐making and the types of messages they are receiving from popular media.
Who wants to know if their care is rationed? Views of citizens and service informants
Authors: Coast Joanna
Published: March 2003

Objective To explore the views of citizens and service informants about whether they would want to know about any rationing of their own health care.

Design In‐depth interviews using a semistructured schedule. Data were analysed using the methods of constant comparison.

Setting and participants Citizens and service informants.

Main variables studied Issues around health care rationing were explored within the context of the United Kingdom health care system.

Results The views of citizens and service informants were very similar in terms of whether they, personally, wanted to know about any rationing of their own care, with the vast majority wanting to be given this information. Informants were also similar in terms of their reasons for wanting to know about rationing: to be given a ‘good explanation’ to enable them to judge whether the decision made had been correct; and to enable them to change the decision if necessary, either through protest or payment. Many informants suggested that they would indeed be likely to react either by challenging the decision or by paying for care.

Conclusions The findings suggest that policies to be open with people about the rationing of care would be welcomed, but also indicate that if protest follows such openness, it may be difficult for the health service to cope with greater explicitness. Further research is needed among patient groups actually facing this situation.
From local concern to randomized trial: the Watcombe Housing Project
Authors: Somerville Margaret , Basham Meryl , Foy Chris , Ballinger Gary , Gay Trevor , Barton Andrew G. ,   on behalf of the Torbay Healthy Housing Group
Published: March 2003

Background  A randomized study of the effect on people's health of improving their housing is underway in Torbay. The link between poor health, particularly respiratory health, and poor housing conditions has been recognized for a long time, but there have been few intervention studies to demonstrate that improving housing can improve health. In 1994, South and West Devon Health Authority set up a community development project in a deprived area of Torbay, in response to the concerns of local primary health‐care workers. A community development worker helped local residents survey their homes for dampness and record their respiratory symptoms. The survey reported high levels of condensation/dampness and respiratory illness and the Council agreed to direct the majority of their housing improvement funds to the estate over the next 3 years. The Health Authority, University of Plymouth and Torbay Council were successful in obtaining funding to evaluate the housing improvements from the NHS R & D programme.

Participants and methods  Of 119 houses eligible for the study, 50 were chosen at random and improved in the first year. The rest were improved the following year. Questionnaires screening for health problems were sent to all 580 residents and baseline surveys of the indoor environment were also carried out. More detailed health surveys were completed by community nurses visiting residents in their homes. All adults were asked to complete SF‐36 and GHQ 12 questionnaires, as well as disease‐specific questionnaires if appropriate.

Progress  All houses in the study have now been improved, including insulation, double‐glazing, re‐roofing, heating, ventilation and electrical rewiring. Follow‐up surveys are underway.
Information and support for women following the primary treatment of breast cancer
Authors: Raupach Jane C. A. , Hiller Janet E.
Published: March 2003

Objective  To examine the need for, use of and satisfaction with information and support following primary treatment of breast cancer.

Design  Cross‐sectional survey.

Participants  Cohort of 266 surviving women diagnosed with breast cancer over a 25‐month period at a tertiary hospital, Adelaide, Australia. Time since diagnosis ranged from 6 to 30 months.

Main outcome measures  Need for, use of and satisfaction with information and support.

Results  Women reported high levels of need for information about a variety of issues following breast cancer treatment. Ninety‐four percentage reported a high level of need for information about one or more issues, particularly recognizing a recurrence, chances of cure and risk to family members of breast cancer. However, few women (2–32%) reported receiving such information. The most frequently used source of information was the surgeon followed by television, newspapers and books. The most frequently used source of support was family followed by friends and the surgeon. Few women (<7%) used formal support services or the Internet. Women were very satisfied with the information and support that they received from the surgeon and other health professionals but reported receiving decreasing amounts of information and support from them over time.

Conclusions  Women experience a high need for information about breast cancer related issues following primary treatment of breast cancer. These needs remain largely unmet as few women receive information about issues that concern them. The role of the surgeon and other health professionals is critical in narrowing the gap between needing and receiving information.
Novel regulatory regions found downstream of the rat B29/Ig‐β gene
Authors: Komatsu Ayano , Otsuka Akira , Ono Masao
Published: March 2003

To search for novel regulatory regions, we examined the features of chromatin structure in the rat B29/Ig‐β gene and its flanking regions by determining DNase I hypersensitive sites (DHS) in plasmacytoma‐derived Y3 cells. Six Y3 cell‐specific DHS were detected at −8.6, promoter, +0.7, +4.4, +6.0, and +8.7 kb. The DHS at +4.4, +6.0, and +8.7 kb were present in the intergenic region between B29/Ig‐β and growth hormone (GH) genes and were mapped inside conserved sequences in rat and humans. In transient transfection into Y3 cells, 2.9‐kb DNA containing the +4.4 and +6.0‐kb DHS demonstrated six times more enhancing activity than B29/Ig‐β promoter alone. Three intergenic DHS each possessed enhancing activity that was highest in the +4.4‐kb region. In the electrophoretic mobility shift assay, a major band shift was demonstrated with Y3 nuclear extract and 0.3‐kb DNA containing the +4.4‐kb region with a conserved 0.22‐kb sequence. By footprint analysis, 20 bases in the middle of the 0.3‐kb DNA were protected by Y3 nuclear extract in which the consensus binding site for the OCT family was present. Deletion of the footprinted region reduced enhancing activity to that of the B29/Ig‐β promoter alone. The sequence responsible for the major band shift and transcriptional enhancing activity in the conserved +4.4‐kb region thus coincided with the 20‐bp footprinted region.
Kinetic properties of bifunctional 6‐phosphofructo‐2‐kinase/ fructose‐2,6‐bisphosphatase from spinach leaves
Authors: Markham Jonathan E. , Kruger Nicholas J.
Published: March 2003

A cDNA encoding 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. The soluble protein had a molecular mass of 320 kDa, estimated by gel filtration chromatography, and␣a␣subunit size of 90.8 kDa. The purified protein had␣activity of␣both 6‐phosphofructo‐2‐kinase (specific␣activity 10.4–15.9 nmol·min−1·mg protein−1) and fructose‐2,6‐bisphosphatase (specific activity 1.65–1.75 nmol·min−1·mg protein−1). The 6‐phosphofructo‐2‐kinase activity was activated by inorganic phosphate, and inhibited by 3‐carbon phosphorylated metabolites and pyrophosphate. In the presence of phosphate, 3‐phosphoglycerate was a mixed inhibitor with respect to both fructose 6‐phosphate and ATP. Fructose‐2,6‐bisphosphatase activity was sensitive to product inhibition; inhibition by inorganic phosphate was uncompetitive, whereas inhibition by fructose 6‐phosphate was mixed. These kinetic properties support the view that the level of fructose 2,6‐bisphosphate in leaves is determined by the relative concentrations of hexose phosphates, three‐carbon phosphate esters and inorganic phosphate in the cytosol through reciprocal modulation of 6‐phosphofructo‐2‐kinase and fructose‐2,6‐bisphosphatase activities of the bifunctional enzyme.
Electrostatic properties of the structure of the docking and dimerization domain of protein kinase A IIα
Authors: Morikis Dimitrios , Roy Melinda , Newlon Marceen G. , Scott John D. , Jennings Patricia A.
Published: March 2003

The structure of the N‐terminal docking and dimerization domain of the type IIα regulatory subunit (RIIα D/D) of protein kinase A (PKA) forms a noncovalent stand‐alone X‐type four‐helix bundle structural motif, consisting of two helix‐loop‐helix monomers. RIIα D/D possesses a strong hydrophobic core and two distinct, exposed faces. A hydrophobic face with a groove is the site of protein–protein interactions necessary for subcellular localization. A highly charged face, opposite to the former, may be involved in regulation of protein–protein interactions as a result of changes in phosphorylation state of the regulatory subunit. Although recent studies have addressed the hydrophobic character of packing of RIIα D/D and revealed the function of the hydrophobic face as the binding site to A‐kinase anchoring proteins (AKAPs), little attention has been paid to the charges involved in structure and function. To examine the electrostatic character of the structure of RIIα D/D we have predicted mean apparent pKa values, based on Poisson–Boltzmann electrostatic calculations, using an ensemble of calculated dimer structures. We propose that the helix promoting sequence Glu34‐X‐X‐X‐Arg38 stabilizes the second helix of each monomer, through the formation of a (i, i +4) side chain salt bridge. We show that a weak inter‐helical hydrogen bond between Tyr35–Glu19 of each monomer contributes to tertiary packing and may be responsible for discriminating from alternative quaternary packing of the two monomers. We also show that an inter‐monomer hydrogen bond between Asp30–Arg40 contributes to quaternary packing. We propose that the charged face comprising of Asp27‐Asp30‐Glu34‐Arg38‐Arg40‐Glu41‐Arg43‐Arg44 may be necessary to provide flexibility or stability in the region between the C‐terminus and the interdomain/autoinhibitory sequence of RIIα, depending on the activation state of PKA. We also discuss the structural requirements necessary for the formation of a stacked (rather than intertwined) dimer, which has consequences for the orientation of the functionally important and distinct faces.
Exploring the role of a glycine cluster in cold adaptation of an alkaline phosphatase
Authors: Mavromatis Konstantinos , Tsigos Iason , Tzanodaskalaki Maria , Kokkinidis Michael , Bouriotis Vassilis
Published: March 2003

In an effort to explore the role of glycine clusters on the cold adaptation of enzymes, we designed point mutations aiming to alter the distribution of glycine residues close to the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Gly261 and Gly262. The replacement of Gly262 by Ala resulted in an inactive enzyme. Substitution of Gly261 by Ala resulted to an enzyme with lower stability and increased energy of activation. The double mutant G261A/Y269A designed on the basis of side‐chain packing criteria from a modelled structure of the enzyme resulted in restoration of the energy of activation to the levels of the native enzyme and in an increased stability compared to the mutant G261A. It seems therefore, that the Gly cluster in combination with its structural environment plays a significant role in the cold adaptation of the enzyme.
Role of sulfoquinovosyl diacylglycerol for the maintenance of photosystem II in Chlamydomonas reinhardtii
Authors: Minoda Ayumi , Sato Norihiro , Nozaki Hisayoshi , Okada Katsuhiko , Takahashi Haruko , Sonoike Kintake , Tsuzuki Mikio
Published: March 2003

The physiological role of sulfoquinovosyl diacylglycerol (SQDG) in photosynthesis was investigated with a SQDG defective mutant (hf‐2) of Chlamydomonas reinhardtii that did not have any detectable amount of SQDG. The mutant showed a lower rate of photosystem II (PSII) activity by ≈␣40% and also a lower growth rate than those of the wild‐type. Results of genetical analysis of hf‐2 strongly suggest that the SQDG defect and the lowered PSII activity are due to a single gene mutation. The supplementation of SQDG to hf‐2 cells restored the lowered PSII activity to the same level as that of wild‐type cells, and also enabled the mutant to grow even in the presence of 135 nm 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea. Moreover, the incubation of isolated thylakoid membranes of hf‐2 with SQDG raised the lowered PSII activity. Chemical modifications of SQDG impaired the recovery of PSII activity. The results suggest that SQDG is indispensable for PSII activity in Chlamydomonas by maintaining PSII complexes in their proper state.
Boar salivary lipocalin
Authors: Spinelli Silvia , Vincent Florence , Pelosi Paolo , Tegoni Mariella , Cambillau Christian
Published: March 2003

The X‐ray structure of variant A of authentic boar salivary lipocalin (SAL), a pheromone‐binding protein specifically expressed in the submaxillary glands of the boar, has been solved and refined at 2.1 Å resolution. The structure displays a classical lipocalin fold with a nine‐stranded sandwiched β barrel and an α helix. A putative glycosylation site, at position 53, has been found to carry a GlcNAc sugar residue. In contrast with what was expected on the basis of mass spectroscopy reports, the internal cavity was found to be devoid of bound pheromonal compound (androstenone or androstenol). Instead, a small electron density volume could be satisfied by a glycerol molecule, a component of the cryoprotecting liquor. The internal cavity was revealed to be very small for steroid compound accommodation. Therefore, docking and molecular dynamics experiments were performed with both pheromonal compounds. These simulations clearly demonstrate a volume increase of the cavity upon steroid binding and the adaptation of the amino‐acid side chains to the steroid molecules. This explains the higher affinity of SAL for both steroid molecules compared to other smaller molecules, although no specific interaction is established with either compound.
Re‐oxygenation of hypoxic simian virus 40 (SV40)‐infected CV1 cells causes distinct changes of SV40 minichromosome‐associated replication proteins
Authors: Riedinger Hans‐Jörg , van Betteraey‐Nikoleit Maria , Probst Hans
Published: March 2003

Hypoxia interrupts the initiation of simian virus 40 (SV40) replication in vivo at a stage situated before unwinding of the␣origin region. After re‐oxygenation, unwinding followed by a synchronous round of viral replication takes place. To␣further characterize the hypoxia‐induced inhibition of unwinding, we analysed the binding of several replication proteins to the viral minichromosome before and after re‐oxygenation. T antigen, the 34‐kDa subunit of replication protein A (RPA), topoisomerase I, the 48‐kDa subunit of primase, the 125‐kDa subunit of polymerase δ, and the 37‐kDa subunit of replication factor C (RFC) were present at the viral chromatin already under hypoxia. The 70‐kDa subunit of RPA, the 180‐kDa subunit of polymerase α, and proliferating cell nuclear antigen (PCNA) were barely detectable at the SV40 chromatin under hypoxia and significantly increased after re‐oxygenation. Immunoprecipitation of minichromosomes with T antigen‐specific antibody and subsequent digestion with micrococcus nuclease revealed that most of the minichromosome‐bound T antigen was associated with the viral origin in hypoxic and in re‐oxygenated cells. T antigen‐catalysed unwinding of the SV40 origin occurred, however, only after re‐oxygenation as indicated by (a) increased sensitivity of re‐oxygenated minichromosomes against digestion with single‐stranded DNA‐specific nuclease P1; (b) stabilization of RPA‐34 binding at the SV40 minichromosome; and (c) additional phosphorylations of RPA‐34 after re‐oxygenation, probably catalysed by DNA‐dependent protein kinase. The results presented suggest that the subunits of the proteins necessary for unwinding, primer synthesis and primer elongation first assemble at the SV40 origin in form of stable, active complexes directly before they start to work.
Recombinant pronapin precursor produced in Pichia pastoris displays structural and immunologic equivalent properties to its mature product isolated from rapeseed
Authors: Palomares Oscar , Monsalve Rafael I. , Rodríguez Rosalía , Villalba Mayte
Published: March 2003

2S albumin storage proteins from rapeseed (Brassica napus), called napins, consist of two different polypeptide chains linked by disulphide bridges, which are derived by proteolytic cleavage from a single precursor. The precursor form of the napin BnIb (proBnIb) has been cloned using a PCR strategy and sequenced. The amino‐acid sequence deduced from the clone includes 31 residues of the small chain and 75 of the large chain, which are connected by the peptide Ser‐Glu‐Asn. Expression of the cDNA encoding proBnIb has been carried out in the methylotrophic yeast Pichia pastoris. The induced protein was secreted to the extracellular medium at a yield of 80 mg·L−1 of culture and was purified by means of size‐exclusion chromatography and reverse phase‐HPLC. Recombinant proBnIb appeared properly folded as its molecular and spectroscopic properties were equivalent to those of the mature heterodimeric protein. As 2S albumin storage proteins from Brassicaceae have been shown to be type I allergy inducers, the immunological activity of the recombinant proBnIb was analysed as a measure of its structural integrity. The immunological properties of the recombinant precursor and the natural napin were indistinguishable by immunoblotting and ELISA inhibition using polyclonal antisera and sera of patients allergic to mustard and rapeseed. In conclusion, the recombinant expression of napin precursors in P. pastoris has been shown to be a successful method for high yield production of homogeneous and properly folded proteins whose polymorphism and complex maturation process limited hitherto their availability.
Human sprouty 4, a new ras antagonist on 5q31, interacts with the dual specificity kinase TESK1
Authors: Leeksma Onno C. , van Achterberg Tanja A. E. , Tsumura Yoshikazu , Toshima Jiro , Eldering Eric , Kroes Wilma G. M. , Mellink Clemens , Spaargaren Marcel , Mizuno Kensaku , Pannekoek Hans , de Vries Carlie J. M.
Published: March 2003

The Drosophila melanogaster protein sprouty is induced upon fibroblast growth factor (FGF)‐ and epidermal growth factor (EGF)‐receptor tyrosine kinase activation and acts as an inhibitor of the ras/MAP kinase pathway downstream of these receptors. By differential display RT‐PCR of activated vs. resting umbilical artery smooth muscle cells (SMCs) we detected a new human sprouty gene, which we designated human sprouty 4 (hspry4) based on its homology with murine sprouty 4. Hspry4 is widely expressed and Northern blots indicate that different isoforms of hspry4 are induced upon cellular activation. The hspry4 gene maps to 5q31.3. It encodes a protein of 322 amino acids, which, in support of a modulating role in signal transduction, contains a prototypic cysteine‐rich region, three, potentially Src homology 3 (SH3) binding, proline‐rich regions and a PEST sequence. This new sprouty orthologue can suppress the insulin‐ and EGF‐receptor transduced MAP kinase signaling pathway, but fails to inhibit MAP kinase activation by constitutively active V12 ras. Hspry4 appears to impair the formation of active GTP‐ras and exert its activity at the level of wild‐type ras or upstream thereof.

 In a yeast two‐hybrid screen, using hspry4 as bait, testicular protein kinase 1 (TESK1) was identified from a human fetal liver cDNA library as a partner of hspry4. The hspry4–TESK1 interaction was confirmed by coimmunoprecipitation experiments and increases by growth factor stimulation. The two proteins colocalize in apparent cytoplasmic vesicles and do not show substantial translocation to the plasma membrane upon receptor tyrosine kinase stimulation.
Reconstitution of Fo of the sodium ion translocating ATP synthase of Propionigenium modestum from its heterologously expressed and purified subunits
Authors: Wehrle Franziska , Appoldt Yvonne , Kaim Georg , Dimroth Peter
Published: March 2003

The atpB and atpF genes of Propionigenium modestum were cloned as His‐tag fusion constructs and expressed in Escherichia coli. Both recombinant subunits a and b were purified via Ni2+ chelate affinity chromatography. A functionally active Fo complex was reassembled in vitro from subunits a, b and c, and incorporated into liposomes. The Fo liposomes catalysed 22Na+ uptake in response to an inside negative potassium diffusion potential, and the uptake was prevented by modification of the c subunits with N,N′‐dicyclohexylcarbodiimide (DCCD). In the absence of a membrane potential the Fo complexes catalysed 22Na+out/Na+in‐exchange. After F1 addition the F1Fo complex was formed and the holoenzyme catalysed ATP synthesis, ATP dependent Na+ pumping, and ATP hydrolysis, which was inhibited by DCCD. Functional Fo hybrids were reconstituted with recombinant subunits a and b from P. modestum and c11 from Ilyobacter tartaricus. These Fo hybrids had Na+ translocation activities that were not distinguishable from that of P. modestum Fo.
Selection of effective antisense oligodeoxynucleotides with a green fluorescent protein‐based assay
Authors: ′t Hoen Peter A. C. , Rosema Bram‐Sieben , Commandeur Jan N. M. , Vermeulen Nico P. E. , Manoharan Muthiah , van Berkel Theo J. C. , Biessen Eric A. L. , Bijsterbosch Martin K.
Published: March 2003

Antisense oligodeoxynucleotides (AS‐ODNs) are frequently used for the down‐regulation of protein expression. Because the majority of potential antisense sequences lacks effectiveness, fast screening methods for the selection of effective AS‐ODNs are needed. We describe a new cellular screening assay for the evaluation of the potency and specificity of new antisense sequences. Fusion constructs of the gene of interest and the gene encoding the enhanced green fluorescent protein (EGFP) are cotransfected with AS‐ODNs to COS‐7 cells. Subsequently, cells are analysed for expression of the EGFP fusion protein by flow cytometry. With the assay, we tested the effectiveness of a set of 15 phosphorothioate ODNs against rat glutathione S‐transferase Mu1 (GSTM1) and/or Mu2 (GSTM2). We found several AS‐ODNs that demonstrated potent, sequence‐specific, and concentration‐dependent inhibition of fusion protein expression. At␣0.5 µm, AS‐6 and AS‐8 inhibited EGFP–GSTM1 expression by 95 ± 4% and 81 ± 6%, respectively. AS‐5 and AS‐10 were selective for GSTM2 (82 ± 4% and 85 ± 0.4% decrease, respectively). AS‐2 and AS‐3, targeted at homologous regions in GSTM1 and GSTM2, inhibited both isoforms (77–95% decrease). Other AS‐ODNs were not effective or displayed non‐target‐specific inhibition of protein expression. The observed decrease in EGFP expression was accompanied by a decrease in GSTM enzyme activity. As isoform‐selective, chemical inhibitors of GSTM and GSTM knock‐out mice are presently unavailable, the selected AS‐ODNs constitute important tools for the study of the role of GSTM in detoxification of xenobiotics and protection against chemical‐induced carcinogenesis.
The role of arginine residues in substrate binding and catalysis by deacetoxycephalosporin C synthase
Authors: Lipscomb Sarah J. , Lee Hwei‐Jen , Mukherji Mridul , Baldwin Jack E. , Schofield Christopher J. , Lloyd Matthew D.
Published: March 2003

Deacetoxycephalosporin C synthase (DAOCS) catalyses the oxidative ring expansion of penicillin N, the committed step in the biosynthesis of cephamycin C by Streptomyces clavuligerus. Site‐directed mutagenesis was used to investigate the seven Arg residues for activity (74, 75, 160, 162, 266, 306 and 307), selected on the basis of the DAOCS crystal structure. Greater than 95% of activity was lost upon mutation of Arg‐160 and Arg266 to glutamine or other residues. These results are consistent with the proposed roles for these residues in binding the carboxylate linked to the nucleus of penicillin N (Arg160 and Arg162) and the carboxylate of the α‐aminoadipoyl side‐chain (Arg266). The results for mutation of Arg74 and Arg75 indicate that these residues play a less important role in catalysis/binding. Together with previous work, the mutation results for Arg306 and Arg307 indicate that modification of the C‐terminus may be profitable with respect to altering the penicillin side‐chain selectivity of DAOCS.
Isolation and characterization of MUC15, a novel cell membrane‐associated mucin
Authors: Pallesen Lone T. , Berglund Lars , Rasmussen Lone K. , Petersen Torben E. , Rasmussen Jan T.
Published: March 2003

The present work reports isolation and characterization of a highly glycosylated protein from bovine milk fat globule membranes, known as PAS III. Partial amino‐acid sequencing of the purified protein allowed construction of degenerate oligonucleotide primers, enabling isolation of a full‐length cDNA encoding a protein of 330 amino‐acid residues. N‐terminal amino‐acid sequencing of derived peptides and the purified protein confirmed 76% of the sequence and demonstrated presence of a cleavable signal peptide of 23 residues, leaving a mature protein of 307 amino acids. Database searches showed no homology to any other proteins. A survey of the human genome indicated the presence of a corresponding gene on chromosome band 11p14.3. Isolation and sequencing of the complete cDNA sequence of the human homologue proved the existence of the gene product (334 amino‐acid residues). This novel mucin‐like protein was named MUC15 by appointment of the HUGO Gene Nomenclature Committee. The deduced amino‐acid sequences of human and bovine MUC15 demonstrated structural hallmarks characteristic for other membrane‐bound mucins, such as a serine, threonine, and proline‐rich extracellular region with several potential glycosylation sites, a putative transmembrane domain, and a short cytoplasmic C‐terminal. We have shown the presence of O‐glycosylations, identified N‐glycosylations at 11 of 15 potential sites in bovine MUC15, and a splice variant encoding a short secreted mucin. Finally, analysis of human and bovine cDNA panels and libraries showed MUC15 gene expression in adult human spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocyte, bone marrow, lymph node, tonsil, breast, fetal liver, bovine lymph nodes and lungs of both species.
Potential active‐site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site‐directed mutagenesis
Authors: Mattern‐Dogru Emine , Ma Xueyan , Hartmann Joachim , Decker Heinz , Stöckigt Joachim
Published: March 2003

In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi‐vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the α/β hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By site‐directed mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model of PNAE was constructed based on the X‐ray crystallographic structure of hydroxynitrile lyase. The potential active site residues were selected on this model, and were mutated in order to better understand the relationship of PNAE with the α/β hydrolases, and as well its mechanism of action. The results showed that PNAE is a novel member of the α/β hydrolase enzyme superfamily.
Suppression of apolipoprotein C‐II amyloid formation by the extracellular chaperone, clusterin
Authors: Hatters Danny M. , Wilson Mark R. , Easterbrook‐Smith Simon B. , Howlett Geoffrey J.
Published: March 2003

The effect of the extracellular chaperone, clusterin, on amyloid fibril formation by lipid‐free human apolipoprotein C‐II (apoC‐II) was investigated. Sub‐stoichiometric levels of clusterin, derived from either plasma or semen, potently inhibit amyloid formation by apoC‐II. Inhibition is dependent on apoC‐II concentration, with more effective inhibition by clusterin observed at lower concentrations of apoC‐II. The average sedimentation coefficient of apoC‐II fibrils formed from apoC‐II (0.3 mg·mL−1) is reduced by coincubation with clusterin (10 µg·mL−1). In contrast, addition of clusterin (0.1 mg·mL−1) to preformed apoC‐II amyloid fibrils (0.3 mg·mL−1) does not affect the size distribution after 2 days. This sedimentation velocity data suggests that clusterin inhibits fibril growth but does not promote fibril dissociation. Electron micrographs indicate similar morphologies for amyloid fibrils formed in the presence or absence of clusterin. The substoichiometric nature of the inhibition suggests that clusterin interacts with transient amyloid nuclei leading to dissociation of the monomeric subunits. We propose a general role for clusterin in suppressing the growth of extracellular amyloid.
Refolding of the Escherichia coli expressed extracellular domain of α7 nicotinic acetylcholine receptor
Authors: Tsetlin Victor I. , Dergousova Natalia I. , Azeeva Ekaterina A. , Kryukova Elena V. , Kudelina Irina A. , Shibanova Elena D. , Kasheverov Igor E. , Methfessel Christoph
Published: March 2003

Heterologous expression of the extracellular domains (ECDs) of the nicotinic acetylcholine receptor (AChR) subunits may give large amounts of proteins for studying the functional and spatial characteristics of their ligand‐binding sites. The ECD of the α7 subunit of the homo‐oligomeric α7 neuronal AChR appears to be a more suitable object than the ECDs of other heteromeric neuronal or muscle‐type AChRs. The rat α7 ECDs (amino‐acid residues ≈ 1–210) were recently expressed in Escherichia coli as fusion proteins with maltose‐binding protein [Fischer, M., Corringer, P., Schott, K., Bacher, A. & Changeux, J. (2001) Proc. Natl Acad. Sci. USA98, 3567–3570] and glutathione S‐transferase (GST) [Utkin, Y., Kukhtina, V., Kryukova, E., Chiodini, F., Bertrand, D., Methfessel, C. & Tsetlin, V. (2001) J. Biol. Chem.276, 15810–15815]. However, these proteins exist in solution mostly as high‐molecular mass aggregates rather than monomers or oligomers. In the present work it is found that refolding of GST–α7‐(1–208) protein in the presence of 0.1% SDS considerably decreases the formation of high‐molecular mass aggregates. The C116S mutation in the α7 moiety was found to further decrease the aggregation and to increase the stability of protein solutions. This mutation slightly increased the affinity of the protein for α‐bungarotoxin (from Kd≈ 300 to 150 nm). Gel‐permeation HPLC was used to isolate the monomeric form of the GST–α7‐(1–208) protein and its mutant almost devoid of SDS. CD spectra revealed that the C116S mutation considerably increased the content of β structure and made it more stable under different conditions. The monomeric C116S mutant appears promising both for further structural studies and as a starting material for preparing the α7 ECD in an oligomeric form.
LRP130, a protein containing nine pentatricopeptide repeat motifs, interacts with a single‐stranded cytosine‐rich sequence of mouse hypervariable minisatellite Pc‐1
Authors: Tsuchiya Naoto , Fukuda Hirokazu , Sugimura Takashi , Nagao Minako , Nakagama Hitoshi
Published: March 2003

Recently, we have identified and purified minisatellite DNA binding proteins (MNBPs) that bind to the mouse hypervariable minisatellite Pc‐1, from NIH3T3 cells. This study describes the isolation and characterization of a mouse leucine‐rich protein (mLRP130) as one of the MNBPs that binds to the C‐rich strand of Pc‐1. The mLRP130 cDNA was demonstrated to encode a polypeptide of 1306 amino‐acid residues with a deduced molecular mass of 137 kDa, and the mLRP130 mRNA is detected in various organs, including heart, brain, liver, skeletal muscle, kidneys and testes. The mLRP130 protein has nine copies of pentatricopeptide repeat (PPR) motifs that are considered to serve as protein–protein interactions. Two forms of the mLRP130 protein were detected in NIH3T3 cells with an approximate molecular mass of 140 kDa (mLRP130) and 100 kDa (mLRP130der), and were detected mainly in nuclear and cytoplasmic fractions, respectively. Immunofluorescence microscopic analysis demonstrated dominant localization of mLRP130 at the perinuclear region, and also in the nucleus and cytoplasm with dot‐ or squiggle‐like staining. The immunoprecipitated mLRP130 bound to the single‐stranded d(CTGCC)8, but not to its complementary G‐rich strand of d(GGCAG)8 or double‐stranded form. Possible biological roles of mLRP130 are discussed in association with the stability of minisatellite DNA sequences.
The structure of the O‐chain of the lipopolysaccharide of a prototypal diarrheagenic strain of Hafnia alvei that has characteristics of a new species under the genus Escherichia
Authors: Eserstam Reine , Rajaguru Thushari P. , Jansson Per‐Erik , Weintraub Andrej , Albert M. John
Published: March 2003

The structure of the O‐polysaccharide of the lipopolysaccharide from a diarrheal strain isolated in Bangladesh was studied with sugar, and methylation analysis, NMR spectroscopy, mass spectrometry and partial acid hydrolysis. The strain was first designated as Hafnia alvei, but later found to be a possible new species in the genus Escherichia. Two different polysaccharides were detected, a major and a minor one. The structure of the major polysaccharide is given below, while the structure of the minor one was not investigated. The structure of the repeating unit was established as

The structure does not resemble any of the previously investigated lipopolysaccharide O‐chains from Escherichia coli or H. alvei, but could fit in either group based on types of sugar residues and acidity.

Phenotypic microbiological studies cannot definitely assign it to either species of the two genera. Genetic hybridization studies indicate that the Bangladeshi isolates may require a new species designation under the genus Escherichia.