bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of proxies for European and North Atlantic temperature field reconstructions
Authors: Pauling A. , Luterbacher J. , Wanner H.
Published: August 2003

We evaluate the importance of high‐resolution proxies for boreal winter (October to March) and summer (April to September) European and North Atlantic temperature reconstructions. Multiple regression, backward elimination and cross‐validation techniques are used to achieve this goal. The analysis considers natural proxies and synthetic “pseudo‐documentary indices”. The results suggest that the most valuable predictors for European winter temperature are documentary‐based indices, while tree‐rings performed best for the warm season. It was also shown that the temperature signal in a speleothem from Scotland may be used for further winter and summer temperature reconstructions over parts of the Atlantic Ocean. This study represents a step towards the optimal selection of proxies which will improve temperature reconstructions.
Size and density sorting of metal and silicate grains under microgravity conditions and the origin of chondrites
Authors: Franzen M. A. , Nichols S. , Bogdon K. , White C. , Godsey R. , Napieralski N. , Benoit P. H. , Sears D. W. G.
Published: August 2003

We report here experiments on NASA's KC‐135 microgravity facility aimed at investigating metal‐silicate fractionation in chondrites. Metal and sand particle mixtures, with sizes and relative proportions approximating chondritic values, were allowed to undergo density and aerodynamic sorting as air was passed through 310, ten‐centimeter long 2.54 cm diameter columns under microgravity conditions. Metal and silicate fractionation was found to occur when gases passed through the mixtures and when the particle diameter ratio was large (∼3.0), with sand being enriched near the surface of the beds. This is contrary to experiments performed under terrestrial gravity and the semi‐empirical Ergun equation. While the reason for this discrepancy is not well understood, the present results do suggest that separation of metal and silicates will readily occur under microgravity conditions on the surface of asteroids.
Atmospheric response to solar radiation absorbed by phytoplankton
Authors: Shell K. M. , Frouin R. , Nakamoto S. , Somerville R. C. J.
Published: August 2003

Phytoplankton alter the absorption of solar radiation, affecting upper ocean temperature and circulation. These changes, in turn, influence the atmosphere through modification of the sea surface temperature (SST). To investigate the effects of the present‐day phytoplankton concentration on the atmosphere, an atmospheric general circulation model was forced by SST changes due to phytoplankton. The modified SST was obtained from ocean general circulation model runs with space‐ and time‐varying phytoplankton abundances from Coastal Zone Color Scanner data. The atmospheric simulations indicate that phytoplankton amplify the seasonal cycle of the lowest atmospheric layer temperature. This amplification has an average magnitude of 0.3°K but may reach over 1°K locally. The surface warming in the summer is marginally larger than the cooling in the winter, so that on average annually and globally, phytoplankton warm the lowest layer by about 0.05°K. Over the ocean the surface air temperature changes closely follow the SST changes. Significant, often amplified, temperature changes also occur over land. The climatic effect of phytoplankton extends throughout the troposphere, especially in middle latitudes where increased subsidence during summer traps heat. The amplification of the seasonal cycle of air temperature strengthens tropical convection in the summer hemisphere. In the eastern tropical Pacific Ocean a decreased SST strengthens the Walker circulation and weakens the Hadley circulation. These significant atmospheric changes indicate that the radiative effects of phytoplankton should not be overlooked in studies of climate change.
Changes in Lena River streamflow hydrology: Human impacts versus natural variations
Authors: Ye Baisheng , Yang Daqing , Kane Douglas L.
Published: August 2003

This study systematically analyzes long‐term (1936–1999) monthly discharge records for the major subbasins within the Lena River watershed in order to document significant streamflow hydrology changes induced by human activities (particularly reservoirs) and by natural variations/changes. The results show that the upper streams of the watershed, without much human impact, experience a runoff increase in winter, spring, and (particularly) summer seasons and a discharge decrease in fall season. These changes in seasonal streamflow characteristics indicate a hydrologic regime shift toward early snowmelt and higher summer streamflow perhaps due to regional climate warming and permafrost degradation in the southern parts of Siberia. The results also demonstrate that reservoir regulations have significantly altered the monthly discharge regime in the lower parts of Lena River basin. Because of a large dam in west Lena River, summer (high) flows at the outlet of the Vilui valley have been reduced by up to 55% and winter (low) flows have been increased by up to 30 times. These alterations, plus streamflow changes in the upper Lena regions, lead to strong upward trends (up to 90%) in monthly discharge at the basin outlet during the low‐flow months and weak increases (5–10%) in the high‐flow season. Monthly flow records at the basin outlet have been reconstructed by a regression method to reduce reservoir impacts. Trend analyses and comparisons between the observed and reconstructed monthly flows show that because of reservoir regulations, discharge records observed at the Lena basin outlet do not always represent natural changes and variations. They tend to underestimate the natural runoff trends in summer and overestimate the trends in both winter and fall seasons. Therefore the cold season discharge increase identified at the mouth of the Lena basin is not all naturally caused, but the combined effect of reservoir regulation and natural runoff changes in the unregulated upper subbasins. This study clearly illustrates the importance of human activities in regional and global environment changes and points to a need to examine human impacts in other large high‐latitude watersheds.
Levocetirizine is effective for symptom relief including nasal congestion in adolescent and adult (PAR) sensitized to house dust mites
Authors: Potter P. C. ,   on behalf of the Study Group
Published: August 2003

Background: Antihistamines are the most commonly prescribed class of medication for perennial allergic rhinitis (PAR). The primary objective of this study was to determine whether levocetirizine (Xyzal®), the active enantiomer of cetirizine, could achieve at least a 50% improvement in PAR symptoms compared to the placebo over the first week of treatment.

Methods: A total of 294 patients with PAR due to house dust mites were randomized in this 8‐week double‐blind, placebo‐controlled, multicentre trial to receive either levocetirizine 5 mg/day or placebo. Mean Total Four‐Symptom Scores (T4SS) (nasal pruritus, ocular pruritus, rhinorrhoea and sneezing) were compared between treatment groups over weeks 1, 4 and 6. All individual symptom scores, including nasal congestion, were also studied.

Results: Levocetirizine showed an 86% improvement in T4SS over the first week of treatment and a 47% improvement over the entire treatment period compared with placebo. Absolute changes from baseline were 3.64 and 2.47 for levocetirizine and placebo, respectively. Individual symptom scores showed statistically significant (P ≤ 0.01) differences in favour of levocetirizine for all study time‐points. Nasal congestion was unexpectedly significantly improved (P < 0.001). The incidence of reported adverse events was comparable between treatment and placebo group.

Conclusions: Levocetirizine 5 mg/day is an effective and well‐tolerated treatment of PAR. In addition, levocetirizine is effective for the relief of nasal congestio.
Effect of anti‐nerve growth factor on early and late airway responses in allergic rats
Authors: Glaab T. , Hoymann H. G. , Hecht M. , Korolewitz R. , Tschernig T. , Hohlfeld J. M. , Krug N. , Braun A.
Published: August 2003

Background: The increased production of nerve growth factor (NGF) has been associated with allergen‐induced airway hyperresponsiveness and enhanced airway inflammation in experimental models of asthma. The aim of this study was to investigate whether a local application of anti‐NGF to the lungs may affect the allergen‐specific early (EAR) and late (LAR) airway responses to ovalbumin (Ova) of Ova‐sensitized brown Norway rats.

Methods: Rats were sensitized systemically with Ova and were boosted twice intratracheally with Ova aerosol using a microsprayer. Two hours before every boost, the animals were pretreated either with aerosolized anti‐NGF or with a control antibody. On day 21, all animals were challenged with inhalational Ova aerosol and pulmonary resistance was recorded in anesthetized, orotracheally intubated animals during the early and late asthmatic responses. In addition, differential cell counts from bronchoalveolar lavage and serum immunoglobulin E (IgE) levels were determined 48 h post‐Ova challenge.

Results: Pretreatment with anti‐NGF significantly attenuated the EAR but had no significant effect on the LAR. Serum IgE levels and inflammatory cell influx into the lungs were not affected by anti‐NGF pretreatment.

Conclusion: The data from this study suggest that NGF is directly involved in the development of the EAR without affecting the inflammatory airway response or LAR.
A further evaluation of the clinical use of specific IgE antibody testing in allergic diseases
Authors: Söderström L. , Kober A. , Ahlstedt S. , de Groot H. , Lange C.‐E. , Paganelli R. , Roovers M. H. W. M. , Sastre J.
Published: August 2003

Background: The evaluation and interpretation of the results from blood tests measuring specific immunoglobulin E (IgE) antibody concentration is currently made using the dichotomized result from the test despite a quantitative result is obtained. It has been shown that different levels of IgE antibodies, assessed by blood test and skin prick test, may have a relation to presence of symptoms, implying that there is more information in a quantitative result than in the dichotomous – positive or negative.

Objective: To investigate the clinical utility of quantification of IgE antibodies in the diagnosis of allergic patients and whether such procedure has any advantage to the presently dichotomously used sensitivity and specificity at a fixed cut‐off.

Methods: Data from a previously published study (R. Paganelli, I.J. Ansoteugi, J. Sastre, C.‐E. Lange, M.H.W.M. Roovers, H. de Groot, N.B. Lindholm, P.W. Ewan, Allergy, 1998; 53) analysing diagnosis of allergic patients in four different clinics were re‐evaluated. In the original study consecutive patients with suspected IgE‐mediated allergy had been examined and evaluated according to the clinical routine at each clinic, using case history, physical examination, skin tests and laboratory tests, except the test to be evaluated, and given an ‘doctors’ allergen‐specific diagnosis’ as positive or negative. In the present study the relation between ‘doctors’ allergen‐specific diagnosis’, expressed as pos/neg, and the quantitative levels of specific IgE antibody concentration was analysed using a logistic regression model. This presentation of results was also compared with the more common characteristics of sensitivity and specificity, and also with Receiver–operator characteristics (ROC) curves.

Results: The used logistic model described the relationship between allergen‐specific diagnosis in each study and the levels of IgE antibodies. The shape of the curve illustrated the physicians’ disposition for a positive diagnose in the study, in relation to the specific IgE antibody level. Differences in the shape of the curve was found both between allergens within clinics and between clinics for the same allergen. No association could be demonstrated between prevalence and shape of the curve.

Conclusions: Conventional sensitivity/specificity figures or ROC concepts only use the qualitative statement of whether IgE is present or not. A risk assessment using the quantitative level of IgE antibody to an allergen increases the utility of the information in clinical context compared with a qualitative statement of whether IgE is present or not. The quantification demonstrated the link between specific IgE antibodies and allergic reactions. The use of objective, well performing quantitative tests should help improve diagnostic accuracy and might provide a way for the patient to understand and manage his or her daily situation and risk for reactions.
Detection of specific IgE antibodies in the sera of patients allergic to birch pollen using recombinant allergens Bet v 1, Bet v 2, Bet v 4: evaluation of different IgE reactivity profiles
Authors: Rossi R. E. , Monasterolo G. , Monasterolo S.
Published: August 2003

Background:  Birch pollen is a significant cause of immediate hypersensitivity among susceptible subjects in temperate climates, affecting 5–54% of the population in western Europe. We examined the specific serum IgE antibodies towards recombinant allergens Bet v 1, Bet v 2 and Bet v 4 in birch‐sensitive patients from the province of Cuneo, north‐west Italy.

Methods:  Sera were obtained from 372 patients with symptomatic birch pollen‐induced allergic rhinitis and/or asthma. A subgroup of these patients suffered from oral allergy syndrome after eating apple. Their sera were evaluated for specific IgE against natural birch pollen and apple extract, as well as Bet v 1, Bet v 2 and Bet v 4 using Pharmacia CAP system (Pharmacia, Uppsala, Sweden).

Results:  Of 372 patients 215 (57.80%) had serum‐specific IgE towards Bet v 1. A total of 166 sera (44.62%) contained serum‐specific IgE to Bet v 2, while Bet v 4 IgE reactivity was documented in 35 subjects (9.41%). Moreover, 146 (39.25%) patients were monosensitized to Bet v 1; 96 (25.81%) patients were monosensitized to Bet v 2; only four sera (1.08%) contained specific IgE towards Bet v 4. Thirty‐nine sera (11.02%) did not contain specific IgE to these individual birch pollen allergens. Of course, all 372 sera (100%) had specific IgE against natural birch pollen extract, of which 162 (43.55%) contained specific IgE to apple extract (75.35% of Bet v 1 positive sera).

Conclusion:  In this study we observed that three birch pollen recombinant allergens alone, could sufficiently identify 90% of birch pollen‐sensitive patients. Therefore, for a more precise IgE profile of patients allergic to birch, further purified birch pollen allergens (i.e. Bet v 6, Bet v 7, Bet v 8) will be required.
Breastfeeding and allergic disease: a multidisciplinary review of the literature (1966–2001) on the mode of early feeding in infancy and its impact on later atopic manifestations
Authors: van Odijk J. , Kull I. , Borres M. P. , Brandtzaeg P. , Edberg U. , Hanson L. Å. , Høst A. , Kuitunen M. , Olsen S. F. , Skerfving S. , Sundell J. , Wille S.
Published: August 2003

Background:  Strategies to prevent children from developing allergy have been elaborated on the basis of state‐of‐the‐art reviews of the scientific literature regarding pets and allergies, building dampness and health, and building ventilation and health. A similar multidisciplinary review of infant feeding mode in relation to allergy has not been published previously. Here, the objective is to review the scientific literature regarding the impact of early feeding (breast milk and/or cow's milk and/or formula) on development of atopic disease. The work was performed by a multidisciplinary group of Scandinavian researchers.

Methods:  The search in the literature identified 4323 articles that contained at least one of the exposure and health effect terms. A total of 4191 articles were excluded mainly because they did not contain information on both exposure and health effects. Consequently, 132 studies have been scrutinized by this review group.

Results:  Of the 132 studies selected, 56 were regarded as conclusive. Several factors contributed to the exclusions. The studies considered conclusive by the review group were categorized according to population and study design.

Conclusions:  The review group concluded that breastfeeding seems to protect from the development of atopic disease. The effect appears even stronger in children with atopic heredity. If breast milk is unavailable or insufficient, extensively hydrolysed formulas are preferable to unhydrolysed or partially hydrolysed formulas in terms of the risk of some atopic manifestations.
T cell activation, from atopy to asthma: more a paradox than a paradigm
Authors: El Biaze M. , Boniface S. , Koscher V. , Mamessier E. , Dupuy P. , Milhe F. , Ramadour M. , Vervloet D. , Magnan A.
Published: August 2003

During the last 15 years, it was largely shown that allergic inflammation was orchestrated by activated Th2 lymphocytes, leading to IgE production and eosinophil activation. Indeed, Th2 activation was shown to be necessary to induce allergic sensitization in animal models. In humans, a Th2 skewing was shown in atopic children soon after birth. In asthma, descriptive studies showed that Th2 cells were more numerous in patients than in controls. In addition, during specific allergen stimulation, an increase of Th2 cells was described in most cases. According to this Th2 paradigm, it was proposed that early avoidance of microbial exposure could explain the increase of atopic diseases seen in the last 20 years in developed countries, as the ‘hygiene hypothesis’. Recently, it was proposed that early exposure to lipopolysaccharide (LPS) could be protective against atopic diseases.

However, it is well established that exposure to LPS can induce asthma symptoms, both in animals and humans, although it induces a Th1 inflammatory response. In addition, most infections induce asthma exacerbations and Th1 responses. Recently, some studies have showed that some Th1 cells were present in asthmatic patients, which could be related to bronchial hyperreactivity.

There is therefore an ‘infectious paradox’ in asthma, which contributes to show that the Th2 paradigm is insufficient to explain the whole inflammatory reaction of this disease. We propose that the Th2paradigm is relevant to atopy and inception of asthma albeit a Th1 activation would account at least in part for bronchial hyperreactivity and asthma symptoms.
Effects of dietary glycerol on the expression of pterin carbinolamine dehydratase in the rat
Authors: CONNOLLY Edward , DONLON John
Published: August 2003

Earlier studies have shown that the abundance of hepatic phenyl-alanine hydroxylase (PAH) diminishes to 60% of control values in rats fed with a diet composed of 40% (w/w) glycerol [Guerin, Walsh, Donlon and Kaufman (1998) Int. J. Biochem. Cell Biol. 30, 1047–1054]. In this experimental model, there are corresponding decreases in the hepatic concentrations of both the hydroxylase cofactor, tetrahydrobiopterin, and the nucleotide guanosine triphosphate. We now show that the cytoplasmic activities of hepatic pterin-4a-carbinolamine dehydratase (PCD) are also lower in these animals, by approx. 50% compared with control values. Immunoblotting confirmed a diminution of protein abundance in vivo. PCD also functions as a dimerization cofactor (DCoH) for the hepatocyte nuclear factor 1α (HNF1α) and the relative abundance of PCD/DCoH in the nucleus is also decreased. There is a small reduction in the mRNA levels for PAH and for PCD/DCoH in the glycerol-fed animals. In the kidney, there is also a diminution in the abundance of both PAH and PCD proteins. Hepatic GTP cyclohydrolase I activity was not altered and the abundance of hepatic HNF1α remained unchanged. HNF1α is required for the expression of PAH in the liver and our results support a role for PCD/DCoH, through its interaction with HNF1α, in regulating the expression of PAH.
Structural and thermal stability analysis of Escherichia coli and Alicyclobacillus acidocaldarius thioredoxin revealed a molten globule-like state in thermal denaturation pathway of the proteins: an infrared spectroscopic study
Authors: PEDONE Emilia , BARTOLUCCI Simonetta , ROSSI Mosè , PIERFEDERICI Francesco Maria , SCIRÈ Andrea , CACCIAMANI Tiziana , TANFANI Fabio
Published: August 2003

The structure of thioredoxin from Alicyclobacillus acidocaldarius (previously named Bacillus acidocaldarius) (BacTrx) and from Escherichia coli (E. coli Trx) was studied by Fourier-transform IR spectroscopy. Two mutants of BacTrx [Lys18→Gly (K18G) and Arg82→Glu (R82E)] were also analysed. The data revealed similar secondary structures in all proteins, but BacTrx and its mutants showed a more compact structure than E. coli Trx. In BacTrx and its mutants, the compactness was p2H-dependent. All proteins revealed the existence of a molten globule-like state. At p2H 5.8, the temperature at which this state was detected was higher in BacTrx and decreased in the different proteins in the following order: BacTrx>R82E>K18G>E. coli Trx. At neutral or basic p2H, the molten globule-like state was detected at the same temperature in both BacTrx and R82E, whereas it was found at the same temperature in all p2Hs tested for E. coli Trx. The thermal stability of the proteins was in the following order at all p2Hs tested: BacTrx>R82E>K18G>E. coli Trx, and was lower for each protein at p2H 8.4 than at neutral or acidic p2Hs. The formation of protein aggregates, brought about by thermal denaturation, were observed for BacTrx and K18G at all p2Hs tested, whereas they were present in R82E and E. coli Trx samples only at p2H 5.8. The results indicated that a single mutation might affect the structural properties of a protein, including its propensity to aggregate at high temperatures. The data also indicated a possible application of Fourier-transform IR spectroscopy for assessing molten globule-like states in small proteins.
Biochemical mechanism of action of a diketopiperazine inactivator of plasminogen activator inhibitor-1
Authors: EINHOLM Anja P. , PEDERSEN Katrine E. , WIND Troels , KULIG Paulina , OVERGAARD Michael T. , JENSEN Jan K. , BØDKER Julie S. , CHRISTENSEN Anni , CHARLTON Peter , ANDREASEN Peter A.
Published: August 2003

XR5118 [(3Z,6Z)-6-benzylidine-3-(5-(2-dimethylaminoethyl-thio-))-2-(thienyl)methylene-2,5-dipiperazinedione hydrochloride] can inactivate the anti-proteolytic activity of the serpin plasminogen activator inhibitor-1 (PAI-1), a potential therapeutic target in cancer and cardiovascular diseases. Serpins inhibit their target proteases by the P1 residue of their reactive centre loop (RCL) forming an ester bond with the active-site serine residue of the protease, followed by insertion of the RCL into the serpin's large central β-sheet A. In the present study, we show that the RCL of XR5118-inactivated PAI-1 is inert to reaction with its target proteases and has a decreased susceptibility to non-target proteases, in spite of a generally increased proteolytic susceptibility of specific peptide bonds elsewhere in PAI-1. The properties of XR5118-inactivated PAI-1 were different from those of the so-called latent form of PAI-1. Alanine substitution of several individual residues decreased the susceptibility of PAI-1 to XR5118. The localization of these residues in the three-dimensional structure of PAI-1 suggested that the XR5118-induced inactivating conformational change requires mobility of α-helix F, situated above β-sheet A, and is in agreement with the hypothesis that XR5118 binds laterally to β-sheet A. These results improve our understanding of the unique conformational flexibility of serpins and the biochemical basis for using PAI-1 as a therapeutic target.
Role of activator protein-1 in the down-regulation of the human CYP2J2 gene in hypoxia
Authors: MARDEN Nicole Y. , FIALA-BEER Eva , XIANG Shi-Hua , MURRAY Michael
Published: August 2003

The cytochrome P450 (CYP) 2J2 arachidonic acid epoxygenase gene was down-regulated at a pre-translational level in human hepatoma-derived HepG2 cells incubated in a hypoxic environment; under these conditions, the expression of c-Jun and c-Fos mRNA and protein was increased. The 5′-upstream region of the CYP2J2 gene was isolated by amplification of a 2341 bp fragment and putative regulatory elements that resembled activator protein-1 (AP-1)-like sequences were identified. From transient transfection analysis, c-Jun was found to strongly activate a CYP2J2–luciferase reporter construct, but co-transfection with plasmids encoding c-Fos or c-Fos-related antigens, Fra-1 and −2, abrogated reporter activity. Using a series of deletion-reporter constructs, a c-Jun-responsive module was identified between bp −152 and −50 in CYP2J2: this region contained an AP-1-like element between bp −56 and −63. The capacity of this element to interact directly with c-Jun, but not c-Fos, was confirmed by electromobility-shift assay analysis. Mutagenesis of the −56/−63 element abolished most, but not all, of the activation of CYP2J2 by c-Jun, thus implicating an additional site within the c-Jun-responsive region. The present results establish an important role for c-Jun in the control of CYP2J2 expression in liver cells. Activation of c-Fos expression by hypoxia promotes the formation of c-Jun/c-Fos heterodimers, which decrease the binding of c-Jun to the CYP2J2 upstream region, leading to gene down-regulation.
The transcription factor early growth response factor-1 (EGR-1) promotes apoptosis of neuroblastoma cells
Authors: PIGNATELLI Miguel , LUNA-MEDINA Rosario , PÉREZ-RENDÓN Arturo , SANTOS Angel , PEREZ-CASTILLO Ana
Published: August 2003

Early growth response factor-1 (EGR-1) is an immediate early gene, which is rapidly activated in quiescent cells by mitogens or in postmitotic neurons after depolarization. EGR-1 has been involved in diverse biological functions such as cell growth, differentiation and apoptosis. Here we report that enforced expression of the EGR-1 gene induces apoptosis, as determined by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labelling (TUNEL) analysis, in murine Neuro2A cells. In accordance with this role of EGR-1 in cell death, antisense oligonucleotides increase cell viability in cells cultured in the absence of serum. This apoptotic activity of the EGR-1 appears to be mediated by p73, a member of the p53 family of proteins, since an increase in the amount of p73 is observed in clones stably expressing the EGR-1 protein. We also observed an increase in the transcriptional activity of the mdm2 promoter in cells overexpressing EGR-1, which is paralleled by a marked decrease in the levels of p53 protein, therefore excluding a role of this protein in mediating EGR-1-induced apoptosis. Our results suggest that EGR-1 is an important factor involved in neuronal apoptosis.
Heterologous expression of four glutathione transferase genes genetically linked to a major insecticide-resistance locus from the malaria vector Anopheles gambiae
Authors: ORTELLI Federica , ROSSITER Louise C. , VONTAS John , RANSON Hilary , HEMINGWAY Janet
Published: August 2003

A cluster of eight genes encoding glutathione transferases (GSTs) are located on division 33B of polytene chromosome arm 3R of the African malaria mosquito, Anopheles gambiae. This region of the genome contains a major 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT)-resistance locus, rtd1. These GSTs belong to the insect-specific Epsilon class and share between 22.6 and 65.2% identity at the amino acid level. Two distinct allelic variants of the Epsilon GST, GSTe1, differing at 12 out of 224 amino acid residues, are present in laboratory and field populations of A. gambiae. To investigate the possible role of these GSTs in conferring resistance to the insecticide DDT, both GSTe1 alleles, plus three additional members of this gene cluster, were expressed in Escherichia coli and the recombinant proteins biochemically characterized. The five putative glutathione transferases encoded catalytically active subunits with variable biochemical properties. For example, the two allelic variants of GSTE1-1 encoded proteins with over 100-fold variation in peroxidase activity, while the three remaining GSTs had no detectable peroxidase activity. Only GSTE2-2 was able to metabolize DDT. Western blots using antibodies raised against these GSTs indicated that the expression of GSTE2-2 is elevated in a DDT-resistant strain of A. gambiae.
Zinc-fingers and homeoboxes (ZHX) 2, a novel member of the ZHX family, functions as a transcriptional repressor
Authors: KAWATA Hiroko , YAMADA Kazuya , SHOU Zhangfei , MIZUTANI Tetsuya , YAZAWA Takashi , YOSHINO Miki , SEKIGUCHI Toshio , KAJITANI Takashi , MIYAMOTO Kaoru
Published: August 2003

Zinc-fingers and homeoboxes (ZHX) 1 is a transcription factor that interacts with the activation domain of the A subunit of nuclear factor-Y (NF-YA). Using a yeast two-hybrid system, a novel ubiquitous transcription factor ZHX2 as a ZHX1-interacting protein was cloned. ZHX2 consists of 837 amino acid residues and contains two zinc-finger motifs and five homeodomains (HDs) as well as ZHX1. The mRNA is expressed among various tissues. ZHX2 not only forms a heterodimer with ZHX1, but also forms a homodimer. Moreover, ZHX2 interacts with the activation domain of NF-YA. Further analysis revealed that ZHX2 is a transcriptional repressor that is localized in the nuclei. Since ZHX2 shares a number of properties in common with ZHX1, we conclude that all these come under the ZHX family. The minimal functional domains of ZHX2 were then characterized. The dimerization domain with both ZHX1 and ZHX2 is the region containing HD1, the domain that interacts with NF-YA is the HD1 to HD2 region, the repressor domain is the HD1 to a proline-rich region. Lastly, using an immunoprecipitation assay, we showed that ZHX2 intrinsically interacts with NF-YA in HEK-293 cells and that ZHX2 represses the promoter activity of the cdc25C gene stimulated by NF-Y in Drosophila Schneider line 2 cells. Thus the ZHX family of proteins may participate in the expression of a number of NF-Y-regulated genes via a more organized transcription network.
Extracellular ATP stimulates the early growth response protein 1 (Egr-1) via a protein kinase C-dependent pathway in the human osteoblastic HOBIT cell line
Authors: PINES Alex , ROMANELLO Milena , CESARATTO Laura , DAMANTE Giuseppe , MORO Luigi , D'ANDREA Paola , TELL Gianluca
Published: August 2003

Extracellular nucleotides exert an important role in controlling cell physiology by activating intracellular signalling cascades. Osteoblast HOBIT cells express P2Y1 and P2Y2 G-protein-coupled receptors, and respond to extracellular ATP by increasing cytosolic calcium concentrations. Early growth response protein 1 (Egr-1) is a C2H2-zinc-finger-containing transcriptional regulator responsible for the activation of several genes involved in the control of cell proliferation and apoptosis, and is thought to have a central role in osteoblast biology. We show that ATP treatment of HOBIT cells increases Egr-1 protein levels and binding activity via a mechanism involving a Ca2+-independent protein kinase C isoform. Moreover, hypotonic stress and increased medium turbulence, by inducing ATP release, result in a similar effect on Egr-1. Increased levels of Egr-1 protein expression and activity are achieved at very early times after stimulation (5 min), possibly accounting for a rapid way for changing the osteoblast gene-expression profile. A target gene for Egr-1 that is fundamental in osteoblast physiology, COL1A2, is up-regulated by ATP stimulation of HOBIT cells in a timescale that is compatible with that of Egr-1 activation.
Low tetrahydrobiopterin biosynthetic capacity of human monocytes is caused by exon skipping in 6-pyruvoyl tetrahydropterin synthase
Authors: LEITNER Karin L. , MEYER Martina , LEIMBACHER Walter , PETERBAUER Anja , HOFER Susanne , HEUFLER Christine , MÜLLER Angelika , HELLER Regine , WERNER Ernst R. , THÖNY Beat , WERNER-FELMAYER Gabriele
Published: August 2003

Biosynthesis of (6R)-5,6,7,8-tetrahydro-l-biopterin (H4-biopterin), an essential cofactor for aromatic amino acid hydroxylases and NO synthases, is effectively induced by cytokines in most of the cell types. However, human monocytes/macrophages form only a little H4-biopterin, but release neopterin/7,8-dihydroneopterin instead. Whereas 6-pyruvoyl tetrahydropterin synthase (PTPS) activity, the second enzyme of H4-biopterin biosynthesis, is hardly detectable in these cells, PTPS mRNA levels were comparable with those of cell types containing intact PTPS activity. By screening a THP-1 cDNA library, we identified clones encoding the entire open reading frame (642 bp) as well as clones lacking the 23 bp exon 3, which results in a premature stop codon. Quantification of the two mRNA species in different cell types (blood-derived cells, fibroblasts and endothelial cells) and cell lines showed that the amount of exon-3-containing mRNA is correlated closely to PTPS activity. The ratio of exon-3-containing to exon-3-lacking PTPS mRNA is not affected by differential mRNA stability or nonsense-mediated mRNA decay. THP-1 cells transduced with wild-type PTPS cDNA produced H4-biopterin levels and expressed PTPS activities and protein amounts comparable with those of fibroblasts. We therefore conclude that exon 3 skipping in transcription rather than post-transcriptional mechanisms is a major cause of the low PTPS protein expression observed in human macrophages and related cell types.
Differential role of glutaredoxin and thioredoxin in metabolic oxidative stress-induced activation of apoptosis signal-regulating kinase 1
Authors: SONG Jae J. , LEE Yong J.
Published: August 2003

Redox-sensing molecules such as thioredoxin (TRX) and glutaredoxin (GRX) bind to apoptosis signal-regulating kinase 1 (ASK1) and suppress its activation. Glucose deprivation disrupted the interaction between TRX/GRX and ASK1 and subsequently activated the ASK1–stress-activated protein kinase/extracellular-signal-regulated kinase kinase–c-Jun N-terminal kinase 1 (JNK1) signal-transduction pathway. l-Buthionine-(S,R)-sulphoximine, which decreases intracellular glutathione content, enhanced glucose deprivation-induced activation of JNK1 by promoting the dissociation of TRX, but not GRX, from ASK1. Treatment of cells with exogenous glutathione disulphide ester resulted in the dissociation of GRX, but not TRX, from ASK1 and the subsequent activation of JNK1. Nonetheless, overexpression of calatase, an H2O2 scavenger, inhibited JNK1 activation and cytotoxicity as well as the dissociation of TRX and GRX from ASK1 during combined glucose deprivation and l-buthionine-(S,R)-sulphoximine treatment. Taken together, glucose deprivation-induced metabolic oxidative stress may activate ASK1 through two different pathways: glutathione-dependent GRX–ASK1 and glutathione-independent TRX–ASK1 pathways.
Subsite specificity (S3, S2, S1', S2' and S3') of oligopeptidase B from Trypanosoma cruzi and Trypanosoma brucei using fluorescent quenched peptides: comparative study and identification of specific carboxypeptidase activity
Authors: HEMERLY Jefferson P. , OLIVEIRA Vitor , DEL NERY Elaine , MORTY Rory E. , ANDREWS Norma W. , JULIANO Maria A. , JULIANO Luiz
Published: August 2003

We characterized the extended substrate binding site of recombinant oligopeptidase B enzymes from Trypanosoma cruzi (Tc-OP) and Trypanosoma brucei (Tb-OP), evaluating the specificity of their S3, S2, S1′, S2′ and S3′ subsites. Five series of internally quenched fluorescent peptides based on the substrate Abz-AGGRGAQ-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine] were designed to contain amino acid residues with side chains of a minimum size, and each residue position of this substrate was modified. Synthetic peptides of different lengths derived from the human kininogen sequence were also examined, and peptides of up to 17 amino acids were found to be hydrolysed by Tc-OP and Tb-OP. These two oligopeptidases were essentially arginyl hydrolases, since for all peptides examined the only cleavage site was the Arg–Xaa bond. We also demonstrated that Tc-OP and Tb-OP have a very specific carboxypeptidase activity for basic amino acids, which depends on the presence of at least of a pair of basic amino acids at the C-terminal end of the substrate. The peptide with triple Arg residues (Abz-AGRRRAQ-EDDnp) was an efficient substrate for Tc-OP and Tb-OP: the Arg–Ala peptide bond was cleaved first and then two C-terminal Arg residues were successively removed. The S1′ subsite seems to be an important determinant of the specificity of both enzymes, showing a preference for Tyr, Ser, Thr and Gln as hydrogen donors. The presence of these amino acids at P1′ resulted in substrates that were hydrolysed with Km values in the sub-micromolar range. Taken together, this work supports the view that oligopeptidase B is a specialized protein-processing enzyme with a specific carboxypeptidase activity. Excellent substrates were obtained for Tb-OP and Tc-OP (Abz-AMRRTISQ-EDDnp and Abz-AHKRYSHQ-EDDnp respectively), which were hydrolysed with remarkably high kcat and low Km values.
Interaction of saposin D with membranes: effect of anionic phospholipids and sphingolipids
Authors: CIAFFONI Fiorella , TATTI Massimo , SALVIOLI Rosa , VACCARO Anna Maria
Published: August 2003

Saposin (Sap) D is an endolysosomal protein that, together with three other similar proteins, Sap A, Sap B and Sap C, is involved in the degradation of sphingolipids and, possibly, in the solubilization and transport of gangliosides. We found that Sap D is able to destabilize and disrupt membranes containing each of the three anionic phospholipids most abundant in the acidic endolysosomal compartment, namely lysobisphosphatidic acid (LBPA), phosphatidylinositol (PI) and phosphatidylserine (PS). The breakdown of the membranes, which occurs when the Sap D concentration on the lipid surface reaches a critical value, is a slow process that gives rise to small particles. The Sap D–particle complexes formed in an acidic milieu can be dissociated by an increase in pH, suggesting a dynamic association of Sap D with membranes. The presence of anionic phospholipids is required also for the Sap D-induced perturbation and solubilization of membranes containing a neutral sphingolipid such as ceramide or a ganglioside such as GM1. At appropriate Sap D concentrations Cer and GM1 are solubilized as constituents of small phospholipid particles. Our findings imply that most functions of Sap D are dependent on its interaction with anionic phospholipids, which mediate the Sap D effect on other components of the membrane such as sphingolipids. On consideration of the properties of Sap D we propose that Sap D might have a role in the definition of the structure and function of membranes, such as the intra-endolysosomal membranes, that are rich in anionic phospholipids.
Inhibition of liver trans-sulphuration pathway by propargylglycine mimics gene expression changes found in the mammary gland of weaned lactating rats: role of glutathione
Authors: ZARAGOZÁ Rosa , GARCÍA Concha , RUS A. Diana , PALLARDÓ Federico V. , BARBER Teresa , TORRES Luis , MIRALLES Vicente J. , VIÑA Juan R.
Published: August 2003

In the lactating mammary gland, weaning produces mitochondrial cytochrome c release and nuclear DNA fragmentation, as determined by gel electrophoresis. This is followed by a significant decrease in lactation. Weaning for 2 h produces an early induction of the tumour suppressor/transcription factor p53, whereas the oncoprotein c-Jun and c-Jun N-terminal kinase are elevated after 24 h of weaning when compared with controls. The expression of p21cip1 and p27kip1, cyclin-dependent kinase inhibitors, was significantly higher in weaned rats when compared with control lactating rats. All the changes mentioned above also happen in the lactating mammary gland when propargylglycine, an inhibitor of the liver trans-sulphuration pathway, is administered. This effect is partially reversed by N-acetylcysteine administration. The administration of buthionine sulphoximine, an irreversible inhibitor of γ-glutamylcysteine synthetase, to lactating rats produces a decrease in GSH levels and changes in protein concentrations and gene transcripts similar to those in rats with impaired trans-sulphuration pathway. These data suggest that the inter-tissue flux of GSH is an important mechanism of l-cysteine delivery to the lactating mammary gland and emphasize the importance of this physiological event in maintaining the gene expression required to sustain lactation.
Characterization of the human activator protein-2gamma (AP-2gamma) gene: control of expression by Sp1/Sp3 in breast tumour cells
Authors: HASLETON Mark D. , IBBITT J. Claire , HURST Helen C.
Published: August 2003

The activator protein-2 (AP-2) family of DNA-binding transcription factors are developmentally regulated and also play a role in human neoplasia. In particular, the AP-2γ protein has been shown to be overexpressed in a high percentage of breast tumours. In the present study, we report the complete sequence determination of the human TFAP2C gene encoding the AP-2γ transcription factor plus the mapping of the transcription start site used in breast tumour-derived cells. The 5′-end of the gene lies within a CpG island and transcription is initiated at a single site within a classical initiator motif. We have gone on to investigate why some breast tumour-derived cell lines readily express AP-2γ, whereas others do not, and show that the proximal promoter (+191 to −312) is differentially active in the two cell phenotypes. DNase footprinting led to the identification of three Sp1/Sp3-binding sites within this region, two of which are absolutely required both for promoter function and cell-type-specific activity. By Western blotting a panel of expressing and non-expressing breast tumour lines we show that the latter have higher levels of Sp3. Furthermore, increasing Sp3 levels in AP-2γ-expressing cells led to the repression of AP-2γ promoter activity, particularly when Sp3 inhibitory function was maximized through sumoylation. We propose that differences in the level and activity of Sp3 between breast tumour lines can determine the expression level of their AP-2γ gene.
S3 to S3' subsite specificity of recombinant human cathepsin K and development of selective internally quenched fluorescent substrates
Authors: ALVES Marcio F. M. , PUZER Luciano , COTRIN Simone S. , JULIANO Maria Aparecida , JULIANO Luiz , BRÖMME Dieter , CARMONA Adriana K.
Published: August 2003

We have systematically examined the S3 to S3′ subsite substrate specificity requirements of cathepsin K using internally quenched fluorescent peptides derived from the lead sequence Abz-KLRFSKQ-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine]. We assayed six series of peptides, in which each position except Gln was substituted with various natural amino acids. The results indicated that the S3–S1 subsite requirements are more restricted than those of S1′–S3′. Cathepsin K preferentially accommodates hydrophobic amino acids with aliphatic side chains (Leu, Ile and Val) in the S2 site. Modifications at P1 residues also have a large influence on cathepsin K activity. Positively charged residues (Arg and Lys) represent the best accepted amino acids in this position, although a particular preference for Gly was found as well. Subsite S3 accepted preferentially basic amino acids such as Lys and Arg. A broad range of amino acids was accommodated in the remaining subsites. We further explored the acceptance of a Pro residue in the P2 position by cathepsin K in order to develop specific substrates for the enzyme. Two series of peptides with the general sequences Abz-KXPGSKQ-EDDnp and Abz-KPXGSKQ-EDDnp (where X denotes the position of the amino acid that is altered) were synthesized. The substrates Abz-KPRGSKQ-EDDnp and Abz-KKPGSKQ-EDDnp were cleaved by cathepsin K at the Arg–Gly and Gly–Ser bonds respectively, and have been shown to be specific for cathepsin K when compared with other lysosomal cysteine proteases such as cathepsins L and B and with the aspartyl protease cathepsin D.
X-ray structure of a putative reaction intermediate of 5-aminolaevulinic acid dehydratase
Authors: ERSKINE Peter T. , COATES Leighton , BUTLER Danica , YOUELL James H. , BRINDLEY Amanda A. , WOOD Steve P. , WARREN Martin J. , SHOOLINGIN-JORDAN Peter M. , COOPER Jonathan B.
Published: August 2003

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase, in which the catalytic site of the enzyme is complexed with a putative cyclic intermediate composed of both substrate moieties, has been solved at 0.16 nm (1.6 Å) resolution. The cyclic intermediate is bound covalently to Lys263 with the amino group of the aminomethyl side chain ligated to the active-site zinc ion in a position normally occupied by a catalytic hydroxide ion. The cyclic intermediate is catalytically competent, as shown by its turnover in the presence of added substrate to form porphobilinogen. The findings, combined with those of previous studies, are consistent with a catalytic mechanism in which the C–C bond linking both substrates in the intermediate is formed before the C–N bond.
Genomic identification and biochemical characterization of a second spermidine/spermine N1-acetyltransferase
Authors: CHEN Ying , VUJCIC Slavoljub , LIANG Ping , DIEGELMAN Paula , KRAMER Debora L. , PORTER Carl W.
Published: August 2003

In the polyamine back-conversion pathway, spermine and spermidine are first acetylated by spermidine/spermine N1-acetyl-transferase (SSAT-1) and then oxidized by polyamine oxidase to produce spermidine and putrescine respectively. Herein we apply homology-search methods to identify novel sequences belonging to a second SSAT, SSAT-2, with a chromosomal location at 17p13.1, which is distinct from SSAT-1 at Xp22. Human SSAT-2 cDNA derived from small-cell lung carcinoma was deduced to encode a 170-amino-acid protein having 46% sequence identity and 64% sequence similarity with SSAT-1. When transiently transfected into HEK-293 cells, SSAT-1 decreased spermidine and spermine pools by ≈30%, while, at the same time, significantly increasing putrescine, N1-acetylspermidine, N1-acetylspermine and N1,N12-diacetylspermine pools. By contrast, transfected SSAT-2 had no effect on intracellular polyamine or acetylated polyamine pools. When enzyme activity was assayed on enzyme extracts from transfected cells, both SSAT-1 and SSAT-2 demonstrated much higher acetylating activity than vector-transfected cells. The data suggest that, in intact cells, SSAT-2 may be compartmentalized or it may be inefficient at low intracellular polyamine concentrations. By substituting candidate substrates in the enzyme assay, we determined that SSAT-1 shows the substrate preference norspermidine=spermidine≫spermine>N1-acetylspermine>putrescine, whereas SSAT-2 shows the preference norspermidine>spermidine=spermine≫N1-acetylspermine=putrescine. Analysis of mRNA levels in cell lines and ESTs (expressed sequence tags) from various tissues by digiNorthern (a web-based tool for virtually displaying expression profiles of query genes based on EST sequences) indicated that SSAT-1 tends to be more widely and highly expressed than SSAT-2. While SSAT-1 mRNA was inducible by polyamine analogues in a variety of cell lines, SSAT-2 was not. The existence of an active, but possibly sequestered, SSAT-2 enzyme suggests that, under certain conditions, it may be recruited into basal or perturbed polyamine metabolism.