bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrating species and habitat data for nature conservation in Great Britain: data sources and methods
Authors: Griffiths G. H. , Eversham B. C. , Roy D. B.
Published: March 2003

1.Britain is unusual in the quantity and quality of species and habitat data available, at both national and regional scales. This paper reviews the sources, coverage and quality of these data.

2.Habitat and species data are used by conservation agencies in England, Scotland and Wales for site selection and for monitoring habitat quality. The paper argues, however, that neither habitat data nor species distribution data on their own are sufficient to locate and monitor habitats for nature conservation purposes effectively.

3.Differences in sampling methodologies between habitat and species surveys present methodological difficulties for the development of an integrated monitoring system that uses both types of data. These problems need to be overcome if habitat and species data are to be used more effectively for nature conservation in the wider countryside.

4.A more integrated system based on the concept of biotope occupancy is proposed and discussed. The implementation of the system would assist with understanding those factors that explain observed patterns in species distribution and diversity, thereby helping to improve the effectiveness of policies for nature conservation.
Vegetation gradients in Atlantic Europe: the use of existing phytosociological data in preliminary investigations on the potential effects of climate change on British vegetation
Authors: Duckworth J. C. , Bunce R. G. H. , Malloch A. J. C.
Published: March 2003

This paper aims to demonstrate the use of available vegetation data from the phytosociological literature in preliminary analyses to generate hypotheses regarding vegetation and climate change.
Geographical ecology of South American desert small mammals: consequences of observations at local and regional scales
Authors: Kelt Douglas A. , Marquet Pablo A. , Brown James H.
Published: March 2003

Five major drylands in South American display very low levels of faunal similarity. The remarkable consistency of faunal structure, however, suggests that it may be possible to extend earlier conclusions based only on the mammal fauna of the Altiplano and the Atacama Desert to all of South America's arid lands.
Chance and determinism in the development of isolated communities
Authors: Ward S. A. , Thornton I. W. B.
Published: March 2003

In the colonization of an island by potentially interacting species both the severity of the competition between them and the length of the interval between their arrivals should affect the likelihood of the second species becoming established. Between‐island variation in priority and the interval between colonizations will depend on the dispersal rates of the two species.

We predict that early colonization should be relatively deterministic, whereas later in the colonization process both the sequence of arrivals and the interval between them will be more variable.

Possible instances of both deterministic and stochastic stages in community development are identified in the colonization of the Krakatau islands by zoochorous forest trees and large zoochorous lianes.
The effect of leukotriene‐modifier drugs on aspirin‐induced asthma and rhinitis reactions
Authors: Berges‐Gimeno M. P. , Simon R. A. , Stevenson D. D.
Published: March 2003

Background Leukotrienes (LTs) appear to be crucial mediators of aspirin (ASA)‐induced lower respiratory tract reactions. Therefore, it is logical to assume that leukotriene‐modifier drugs (LTMDs) might block these reactions.

Objective The aim of this study was to determine whether concomitant treatment with LTMDs was associated with a reduction of ASA‐provoked lower respiratory tract reactions in patients with aspirin‐exacerbated respiratory disease (AERD), when compared to AERD patients who were not treated with LTMDs. Secondly, if ASA‐induced lower respiratory tract reactions were prevented in LTMD‐treated patients, was there then a higher prevalence of upper respiratory reactors or, alternatively, a higher prevalence of blocked reactions (‘non‐reactors’) in this group.

Methods Of 271 patients suspected by history of having AERD, 96 were taking cys‐LT receptor antagonists (cys‐LTRAs) and 12 were taking zileuton at the time of oral ASA challenges. A matched control group of 163 patients was not receiving LTMDs. All subjects underwent standard oral ASA challenges. Reactions were classified as follows: classic [naso‐ocular combined with a 20% or > decline in forced expiratory volume of 1 s (FEV1)]; pure lower (20% or > decline in FEV1 without naso‐ocular); partial asthma (naso‐ocular + 15–20% decline in FEV1); upper only (naso‐ocular with < 15% decline in FEV1); negative (no reactions).

Results In patients treated with cys‐LTRAs, there were significant reductions in numbers of patients with ASA‐induced bronchospastic reactions and a concomitant increase in upper respiratory reactors. There were no significant differences in mean provoking doses of ASA or the percent changes in FEV1 values in both groups. In the 12 patients receiving zileuton, no reactions to ASA (16%) were similar to the cys‐LTRA‐treated group (11%) and the control group (15%).

Conclusion During oral ASA challenges, LTMD treatment appeared to shift target organ responses from both upper and lower respiratory tracts to upper tract alone. LTMD blocking of the entire respiratory tract did not appear to occur.
Subcision: a treatment for cellulite
Authors: Hexsel Dóris Maria ,   md, Mazzuco Rosemari ,   md
Published: March 2003

Background Cellulite is a common clinical condition, with few proven effective therapeutic options. Subcision is a surgical technique that is useful in treating advanced degree cellulite. This study was designed to determine the usefulness of the treatment of cellulite by subcision.

Methods From January 1995 to January 1998, 232 female patients, aged 18–52 years, with cellulite on the thighs and buttocks were treated on an outpatient basis by the subcision technique.

Results In the postoperative period, all the patients had pain, bruises, and hemosiderosis. An improvement in the surface depressions was observed and the patients reported a high degree of satisfaction.

Conclusions This outpatient procedure is effective in the correction of surface depressions on the thighs and buttocks, clinically classified as cellulite.
Diversity, distinctiveness and conservation status of the Mediterranean coastal dung beetle assemblage in the Regional Natural Park of the Camargue (France)
Authors: Lobo Jorge Miguel , Lumaret Jean‐Pierre , Jay‐Robert Pierre
Published: March 2003

The Mediterranean region as a whole has the highest dung beetle species richness within Europe. Natural coastal habitats in this region are among those which have suffered severe human disturbance. We studied dung beetle diversity and distinctiveness within one of the most important coastal protected areas in the west Euro‐Mediterranean region (the regional Park of Camargue, southern France) and made comparisons of dung beetle assemblages with other nearby Mediterranean localities, as well as with other coastal protected area (Doñana National Park, Spain). Our finding showed that: (1) The species richness of coastal habitats in the Camargue is low and only grasslands showed a similar level of species richness and abundance to inland habitats of other Mediterranean localities. The unique habitats of the coastal area (beaches, dunes and marshes) are largely colonized by species widely distributed in the hinterland. (2) In spite of their low general distinctiveness, dune and marsh edges are characterized by the occurrence of two rare, vulnerable, specialized and large roller dung beetle species of the genusScarabaeus. As with other Mediterranean localities, current findings suggest a recent decline ofScarabaeuspopulations and the general loss of coastal dung beetle communities in Camargue. (3) The comparison of dung beetle assemblages between the Camargue and Doñana shows that, in spite of the low local dung beetle species richness in the Camargue, the regional dung beetle diversity is similar between both protected areas. Unique historical and geographical factors can explain the convergence in regional diversity as well as the striking divergence in the composition of dung beetle assemblages between both territories.
Who are you, and who are we? Looking through some key words
Authors: Herxheimer Andrew , Goodare Heather
Published: March 2003

The terminology used to describe individuals who come into contact with health services is problematic. Many of the most commonly used words, for example, patient, consumer, user, carry overtones or imply characteristics, which may be misleading or unacceptable to those to whom they are applied.
Investing in early detection versus intensive treatment for breast cancer: a study of the Israeli public priorities
Authors: Shmueli Amir
Published: March 2003

Objective Public opinion has become one of the main inputs in setting priorities, rationing and allocating health resources. The present study focuses on the priorities of the Israeli public in allocating marginal breast cancer funds between early detection in the healthy majority of the population and intensive treatment of the sick minority in need.

Design A sample of 2030 individuals representing the Israeli Jewish urban population aged 45–75 was interviewed in 1993/4. A full sit‐down interview collected information on several health related issues.

Results Sixty‐one per cent chose to direct the funds to early detection efforts and 35% chose to direct the funds to treating the sick. Four per cent of the population could not decide. Higher education and better health are related to choosing early detection; religious observance is related to choosing the treatment option; and men and older persons tend, more than women and younger respondents, to be undecided.

Conclusions Whilst the majority of the population tend to follow a cost‐effectiveness rationale in the marginal use of breast cancer funds, for more than a third of the population efficiency is not thus important, and they adhere to entitlement based upon a need‐equity principle in allocating health resources.
Does provision of an evidence‐based information change public willingness to accept screening tests?
Authors: Domenighetti Gianfranco , Grilli Roberto , Maggi Jenny Rose
Published: March 2003

Objective

To investigate whether the willingness of the general population to undergo a screening test of questionable effectiveness for pancreatic cancer is influenced by the quality and the extent of the information provided.

Design

Randomised study.

Setting

Switzerland.

Participants

Representative sample (N=1000) of the general population aged over 20.

Interventions

Participants were randomly allocated into two groups (N=500 each), with one group to receive basic and the other extended quality of information. The information was presented in two hypothetical scenarios about implicit and explicit benefits and adverse events of the screening test. Response rates were, respectively, 80.2% (N=401) and 93.2% (N=466).

Main outcome measures

Stated willingness to undergo the screening test.

Results

Out of the 401 participants receiving the basic information scenario, 241 (60%) stated their willingness to accept the test, as compared to the 63/466 (13.5%) exposed to the extended one (P < 0.001). After adjusting for respondent characteristics through a logistic regression model, the ‘information effect’, expressed in terms of odds‐ratio (OR), shows that provision of additional information was related to a 91% (OR 0.09; 95CI: 0.07 – 0.13) relative reduction in the likelihood of accepting the screening test.

Conclusion

The quality and the extent of the information provided about the implicit and explicit benefits and adverse events on hypothetical scenarios of a screening test may dramatically change the willingness of people to participate in the testing. This study suggests that provision of full information on the yield of health care interventions plays an important role in protecting the public from being exposed to procedures of questionable effectiveness.
Practical issues in assisting shared decision‐making
Authors: Feldman‐Stewart Deb , Brundage Michael D. , McConnell Beth A. , Mackillop William J.
Published: March 2003

To facilitate treatment decision‐making, one aims to provide information, present it in a way that makes it as easy as possible to understand, and to help the decision‐maker through the cognitive processes that result in a treatment decision. Decision aids aim to accomplish just these goals and this paper identifies practical issues that we have encountered in creating a decision aid for men with early stage prostate cancer. We highlight the results of studies we carried out to provide an empirical basis for the decision aid that we were developing. Several of the studies were designed to identify what information key players (health professionals, patients and family members) thought was important for the decision‐making process. Another investigation studied methodological considerations in identifying important information. The final study focused on presentation issues. These studies, designed to explore what information was considered important, found great variability among both health care professionals involved in treating patients with prostate cancer (urologists, radiation oncologists, nurses in cancer clinics, and radiation technologists) and among the patients, themselves. The studies also showed that not all information contained within a typical category is of equal importance. A methodological study showed that the information that patients deem to be important to their decision depends on whether they are rating the information that could be provided, or questions that could be answered. Finally, presentation studies showed that the various formats used in presenting quantitative information are processed with differing degrees of accuracy and ease. Each of the above results has implications for those creating decision aids; these implications are highlighted.
Five cases, four actors and a moral: lessons from studies of contested treatment decisions
Authors: McIver Shirley , Ham Chris
Published: March 2003

The case of Jaymee Bowen (child B) illustrated the conflict that may arise over treatment decisions in the National Health Service (NHS). This article reviews four further cases involving disagreement between patients and families on the one hand, and health authorities on the other, and a fifth case in which a health authority questioned the treatment decision of a medical specialist. The cases illustrate the rise of consumerism in health care and the challenge for health authorities in weighing the claims of individual patients against the needs of communities. They also demonstrate the increasing role of lawyers and the courts in resolving disputes over treatment decisions. Clinicians were closely involved in all cases, both in recommending treatment options and in serving as independent advisers when disputes arose. The findings presented here indicate that there is a need to strengthen the process of decision‐making in cases of this kind and to make greater use of evidence in informing decisions. In future, decision‐making needs to be characterized by openness, reason giving, an appeals procedure and regulation of the process to ensure that these conditions are met. The funders of health care also need to consider each individual in his or her own right while also using their resources for the benefit of the population as a whole.
A ‘Third Way’ for lay involvement: what evidence so far?
Authors: Pickard Susan , Smith Keri
Published: March 2003

Aims and objectives This article considers evidence regarding lay involvement in the NHS, following the White Paper’s commitment to rebuild public confidence in an NHS ‘accountable to patients and open to the public and shaped by their views’. It looks at two aspects of lay involvement: the lay board member’s involvement in primary care group (PCG) decision‐making and the engagement of the PCG with the wider public.

Methods The paper analyses data from the first sweep of the annual Tracker Survey of a sample of PCGs in England, led by the National Primary Care Research and Development Centre in collaboration with the King’s Fund between September and December 1999. It draws specifically from the postal questionnaires sent to lay members. Firstly, however, it contextualizes this data by reviewing the history of lay involvement before 1997 in the NHS and particularly in primary care.

Conclusions The paper concludes that, during the first 6 months of their operation, the lay voice was faintly heard in PCGs. The lay member’s role in decision‐making at board‐level was peripheral. The majority rated their involvement in key aspects of decision‐making as low and their influence on decision‐making below that of other board members including the Chief Officer, the chair and the GP board members. Beyond the arena of the board, what little contact there was with the lay voice has taken the shape of informing rather than consulting. Mitigating factors include the early stage at which the survey was completed and the lack of precedents for lay involvement in primary care in a broad sense on which PCGs can draw.
Improving the quality of health information: the contribution of C‐H‐i‐Q
Authors: Hain Tom
Published: March 2003

The quality of information should be the key driver in communicating health messages to the public. Staff at The Centre for Health Information Quality (C‐H‐i‐Q) have worked with an extensive list of partner organizations from a range of disciplines. These stakeholders express a desire to see improvement in the standards of patient information, and display a willingness to embrace new approaches in order to achieve change. The result is an increasingly significant contribution in the development of the information‐exchange model of health‐care, where the aim is to maximize public confidence in health information. Key activities include defining principles of quality assurance, engaging with stakeholders, and developing tools and training for producers and providers of health information.
Proteolytic action of duodenase is required to induce DNA synthesis in pulmonary artery fibroblasts
Authors: Pemberton Alan D. , Zamolodchikova Tatyana S. , Scudamore Cheryl L. , Chilvers Edwin R. , Miller Hugh R. P. , Walker Trevor R.
Published: March 2003

Duodenase is a 29‐kDa serine endopeptidase that displays selective trypsin‐ and chymotrypsin‐like substrate specificity. This enzyme has been localized to epitheliocytes of Brunner's glands, and as described here, to mast cells within the intestinal mucosa and lungworm‐infected lung, implying an important additional role in inflammation and tissue remodelling. In primary cultures of pulmonary artery fibroblasts, duodenase induced a concentration‐dependent increase in [3H]thymidine incorporation with a maximal effect observed at 30 nm. Pretreating duodenase with soybean trypsin inhibitor abolished DNA synthesis, confirming that proteolytic activity was an essential requirement for this response. PAR1, PAR2 and PAR4 activating peptides were unable to induce [3H]thymidine incorporation in pulmonary artery fibroblasts. Likewise, pretreatment of fibroblasts with TNFα, known to up‐regulate PAR2 expression in other systems, and IL‐1β, did not enhance the potential of duodenase to induce DNA synthesis. Furthermore, duodenase increased GTPγS binding to fibroblast membranes indicating that a G‐protein‐coupled receptor may mediate the effects of duodenase. Duodenase‐induced DNA synthesis and GTPγS binding were both found to be inhibited by pertussis toxin, implying a role for Gi/o. Selective inhibitors of MEK1 (PD98059) and protein kinase C (GF109203X) only partially inhibited duodenase‐induced DNA synthesis, but both wortmannin (100 nm) and LY294002 (10 µm) inhibited this response completely, indicating a key role for PtdIns 3‐kinase. Furthermore, duodenase induced a 2.3 ± 0.1‐fold increase in PtdIns 3‐kinase activity in p85 immunoprecipitates, which was sensitive to inhibition by wortmannin. These results suggest that duodenase can induce pulmonary artery fibroblast DNA synthesis in a PtdIns 3‐kinase‐dependent manner via a G‐protein‐coupled receptor which is activated by a proteolytic mechanism.
Exploration of the diaphorase activity of neutrophil NADPH oxidase
Authors: Poinas Alexandra , Gaillard Jacques , Vignais Pierre , Doussiere Jacques
Published: March 2003

In the O2– generating flavocytochrome b, the membrane‐bound component of the neutrophil NADPH oxidase, electrons are transported from NADPH to O2 in the following sequence: NADPH→FAD→heme b→O2. Although p‐iodonitrotetrazolium (INT) has frequently been used as a probe of the diaphorase activity of the neutrophil flavocytochrome b, the propensity of its radical to interact reversibly with O2 led us to question its specificity. This study was undertaken to reexamine the interaction of INT with the redox components of the neutrophil flavocytochrome b. Two series of inhibitors were used, namely the flavin analog 5‐deaza FAD and the heme inhibitors bipyridyl and benzylimidazole. The following results indicate that INT reacts preferentially with the hemes rather than with the FAD redox center of flavocytochrome b and is not therefore a specific probe of the diaphorase activity of flavocytochrome b. First, in anaerobiosis, reduced heme b in activated membranes was reoxidized by INT as efficiently as by O2 even in the presence of concentrations of 5‐deaza FAD which fully inhibited the NADPH oxidase activity. Second, the titration curve of dithionite‐reduced heme b in neutrophil membranes obtained by oxidation with increasing amounts of INT was strictly superimposable on that of dithionite‐reduced hemin. Third, INT competitively inhibited the O2 uptake by the activated NADPH oxidase in a cell‐free system. Finally, the heme inhibitor bipyridyl competitively inhibited the reduction of INT in anaerobiosis, and the oxygen uptake in aerobiosis.
Evidence that a eukaryotic‐type serine/threonine protein kinase from Mycobacterium tuberculosis regulates morphological changes associated with cell division
Authors: Chaba Rachna , Raje Manoj , Chakraborti Pradip K.
Published: March 2003

A eukaryotic‐type protein serine/threonine kinase, PknA, was cloned from Mycobacterium tuberculosis strain H37Ra. Sequencing of the clone indicated 100% identity with the published pknA sequence of M. tuberculosis strain H37Rv. PknA fused to maltose‐binding protein was expressed in Escherichia coli; it exhibited a molecular mass of ≈ 97 kDa. The fusion protein was purified from the soluble fraction by affinity chromatography using amylose resin. In vitro kinase assays showed that the autophosphorylating ability of PknA is strictly magnesium/manganese‐dependent, and sodium orthovanadate can inhibit this activity. Phosphoamino‐acid analysis indicated that PknA phosphorylates at serine and threonine residues. PknA was also able to phosphorylate exogenous substrates, such as myelin basic protein and histone. A comparison of the nucleotide‐derived amino‐acid sequence of PknA with that of functionally characterized prokaryotic serine/threonine kinases indicated its possible involvement in cell division/differentiation. Protein–protein interaction studies revealed that PknA is capable of phosphorylating at least a ≈56‐kDa soluble protein from E. coli. Scanning electron microscopy showed that constitutive expression of this kinase resulted in elongation of E. coli cells, supporting its regulatory role in cell division.
Expression pattern in the antennae of a newly isolated lepidopteran Gq protein α subunit cDNA
Authors: Jacquin‐Joly Emmanuelle , François Marie‐Christine , Burnet Michael , Lucas Philippe , Bourrat Franck , Maida Rosario
Published: March 2003

From the antennae of the moth Mamestra brassicae, we have identified a lepidopteran G protein α subunit belonging to the Gq family, through immunological detection in crude antennal extract and antennal primary cell cultures, followed by molecular cloning. The complete cDNA sequence (1540 bp) contains an open reading frame encoding a protein of 353 amino acids. This deduced sequence possesses all of the characteristics of the Gq family and shares a very high degree of amino‐acid sequence identity with vertebrate (80% with mouse or human Gqα) and invertebrate subunits (varying between 60 and 87% for Gqα from organisms as diverse as sponge and Drosophila). The expression pattern of the Gq subunit in adult antennae was associated with the olfactory sensilla suggesting a specific role in olfaction. These data provide molecular evidence for a component of the phosphoinositide signaling pathway in moth antennae: this G protein α subunit may be involved in the olfaction transduction process through interaction with G‐protein‐coupled receptors, stimulating the phospholipase C mediated second messenger pathway.
Purification, characterization, cloning, and expression of the chicken liver ecto‐ATP‐diphosphohydrolase
Authors: Knowles Aileen F. , Nagy Agnes K. , Strobel Randy S. , Wu‐Weis Mae
Published: March 2003

We previously demonstrated that the major ecto‐nucleoside triphosphate phosphohydrolase in the chicken liver membranes is an ecto‐ATP‐diphosphohydrolase (ecto‐ ATPDase) [Caldwell, C., Davis, M.D. & Knowles, A.F. (1999) Arch. Biochem. Biophys. 362, 46–58]. Enzymatic properties of the liver membrane ecto‐ATPDase are similar to those of the chicken oviduct ecto‐ATPDase that we have previously purified and cloned. Using antibody developed against the latter, we have purified the chicken liver ecto‐ATPDase to homogeneity. The purified enzyme is a glycoprotein with a molecular mass of 85 kDa and a specific activity of ≈ 1000 U·mg protein−1. Although slightly larger than the 80‐kDa oviduct enzyme, the two ecto‐ATPDases are nearly identical with respect to their enzymatic properties and mass of the deglycosylated proteins. The primary sequence of the liver ecto‐ATPDase deduced from its cDNA obtained by RT‐PCR cloning also shows only minor differences from that of the oviduct ecto‐ATPDase. Immunochemical staining demonstrates the distribution of the ecto‐ATPDase in the bile canaliculi of the chicken liver. HeLa cells transfected with the chicken liver ecto‐ATPDase cDNA express an ecto‐nucleotidase activity with characteristics similar to the enzyme in its native membranes, most significant of these is stimulation of the ATPDase activity by detergents, which inhibits other members of the ecto‐ nucleoside triphosphate diphosphohydrolase (E‐NTPDase) family. The stimulation of the expressed liver ecto‐ATPDase by detergents indicates that this property is intrinsic to the enzyme protein, and cannot be attributed to the lipid environment of the native membranes. The molecular identification and expression of a liver ecto‐ATPDase, reported here for the first time, will facilitate future investigations into the differences between structure and function of the different E‐NTPDases, existence of liver ecto‐ATPDase isoforms in different species, its alteration in pathogenic conditions, and its physiological function.
Na+/K+‐ATPase as a signal transducer
Authors: Xie Zijian , Askari Amir
Published: March 2003

Na+/K+‐ATPase as an energy transducing ion pump has been studied extensively since its discovery in 1957. Although early findings suggested a role for Na+/K+‐ATPase in regulation of cell growth and expression of various genes, only in recent years the mechanisms through which this plasma membrane enzyme communicates with the nucleus have been studied. This research, carried out mostly on cardiac myocytes, shows that in addition to pumping ions, Na+/K+‐ATPase interacts with neighboring membrane proteins and organized cytosolic cascades of signaling proteins to send messages to the intracellular organelles. The signaling pathways that are rapidly elicited by the interaction of ouabain with Na+/K+‐ATPase, and are independent of changes in intracellular Na+ and K+ concentrations, include activation of Src kinase, transactivation of the epidermal growth factor receptor by Src, activation of Ras and p42/44 mitogen‐activated protein kinases, and increased generation of reactive oxygen species by mitochondria. In cardiac myocytes, the resulting downstream events include the induction of some early response proto‐oncogenes, activation of the transcription factors, activator protein‐1 and nuclear factor kappa‐B, regulation of a number of cardiac growth‐related genes, and stimulation of protein synthesis and myocyte hypertrophy. For these downstream events, the induced reactive oxygen species and rise in intracellular Ca2+ are essential second messengers. In cells other than cardiac myocytes, the proximal pathways linked to Na+/K+‐ATPase through protein–protein interactions are similar to those reported in myocytes, but the downstream events and consequences may be significantly different. The likely extracellular physiological stimuli for the signal transducing function of Na+/K+‐ATPase are the endogenous ouabain‐like hormones, and changes in extracellular K+ concentration.
The N‐terminus of m5C‐DNA methyltransferase MspI is involved in its topoisomerase activity
Authors: Bhattacharya Sanjoy K. , Dubey Ashok K.
Published: March 2003

DNA cytosine methyltransferase MspI (M.MspI) must require a different type of interaction of protein with DNA from other bacterial DNA cytosine methyltransferases (m5C‐MTases) to evoke the topoisomerase activity that it possesses in addition to DNA‐methylation ability. This may require a different structural organization in the solution phase from the reported consensus structural arrangement for m5C‐MTases. Limited proteolysis of M.MspI, however, generates two peptide fragments, a large one (p26) and a small one (p18), consistent with reported m5C‐MTase structures. Examination of the amino‐acid sequence of M.MspI revealed similarity to human topoisomerase I at the N‐terminus. Alignment of the amino‐acid sequence of M.MspI also uncovered similarity (residues 245–287) to the active site of human DNA ligase I. To evaluate the role of the N‐terminus of M.MspI, 2‐hydroxy‐5‐nitrobenzyl bromide (HNBB) was used to truncate M.MspI between residues 34 and 35. The purified HNBB‐truncated protein has a molecular mass of ≈ 45 kDa, retains DNA binding and methyltransferase activity, but does not possess topoisomerase activity. These findings were substantiated using a purified recombinant MspI protein with the N‐terminal 34 amino acids deleted. Changing the N‐terminal residues Trp34 and Tyr74 to alanine results in abolition of the topoisomerase I activity while the methyltransferase activity remains intact.
Cloning of the manganese lipoxygenase gene reveals homology with the lipoxygenase gene family
Authors: Hörnsten Lena , Su Chao , Osbourn Anne E. , Hellman Ulf , Oliw Ernst H.
Published: March 2003

Manganese lipoxygenase was isolated to homogeneity from the take‐all fungus, Gaeumannomyces graminis. The C‐terminal amino acids and several internal peptides were sequenced, and the information was used to obtain a cDNA probe by RT/PCR. Screening of a genomic library of G. graminis yielded a full‐length clone of the Mn‐Lipoxygenase gene. cDNA analysis showed that the gene spanned 2.6 kb and contained one intron (133 bp). Northern blot analyses indicated two transcripts (2.7 and 3.1 kb). The deduced amino‐acid sequence of the Mn‐Lipoxygenase precursor (618 amino acids, 67.7 kDa) could be aligned with mammalian and plant lipoxygenases with 23–28% identity over 350–400 amino‐acid residues of the catalytic domains. Lipoxygenases have one water molecule and five amino acids as Fe ligands. These are two histidine residues in the highly conserved 30 amino‐acid sequence WLLAK‐X15‐H‐X4‐H‐X3‐E of α helix 9, one histidine and usually an asparaine residue in the sequence H‐X3‐N‐X‐G of α helix 18, and the carboxyl oxygen of the C‐terminal isoleucine (or valine) residue. The homologous sequence of α helix 9 of Mn‐Lipoxygenase [WLLAK‐X14‐H(294)‐X3‐H(297)‐X3‐E] contained two single‐amino‐acid gaps, but otherwise His294 and His297 aligned with the two His residues, which coordinate iron. Mn‐Lipoxygenase [H(478)‐X3‐N(482)‐X‐G] could be aligned with the two metal ligands of α helix 18, and the C‐terminal residue was Val618. We conclude that Mn‐Lipoxygenase belongs to the lipoxygenase gene family and that its unique biochemical properties might be related to structural differences in the metal centre and α helix 9 of lipoxygenases rather than to the metal ligands.
Analysis of flux estimates based on 13C‐labelling experiments
Authors: Christensen Bjarke , Karoly Gombert Andreas , Nielsen Jens
Published: March 2003

Modelling of the fluxes in central metabolism can be performed by combining labelling experiments with metabolite balancing. Using this approach, multiple samples from a cultivation of Saccharomyces cerevisiae in metabolic and isotopic steady state were analysed, and the metabolic fluxes in central metabolism were estimated. In the various samples, the estimates of the central metabolic pathways, the tricarboxylic acid cycle, the oxidative pentose phosphate pathway and the anaplerotic pathway, showed an unprecedented reproducibility. The high reproducibility was obtained with fractional labellings of individual carbon atoms as the calculational base, illustrating that the more complex modelling using isotopomers is not necessarily superior with respect to reproducibility of the flux estimates. Based on these results some general difficulties in flux estimation are discussed.
A novel DNA repair enzyme containing RNA recognition, G‐patch and specific splicing factor 45‐like motifs in the protozoan parasite Toxoplasma gondii
Authors: Dendouga Najoua , Callebaut Isabelle , Tomavo Stanislas
Published: March 2003

We report the cloning and functional charaterization of the full‐length cDNA and gene encoding a Toxoplasma gondii DNA repair enzyme designated TgDRE. The gene is composed of three exons separated by two introns of 780 and 630 bp, and encodes a protein with a predicted molecular mass of 49.6 kDa. The native TgDRE protein, with a molecular mass of 60 kDa, is only detected in the virulent tachyzoite stage of T. gondii. However, the transcript is present in both asexual parasite stages, virulent tachyzoite and avirulent encysted bradyzoite. When an Escherichia coli mutant lacking ruvC endonuclease and recG helicase was transformed with TgDRE cDNA, a significant increase in resistance to DNA‐damaging agents, such as UV light and mitomycin C, was observed. Moreover, database searches revealed that TgDRE orthologues were present in the genome sequences of the related apicomplexa parasites Plasmodium falciparum and Plasmodium yoelii, as well as in those of Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans and Homo sapiens. This novel family of proteins is characterized by the presence of human splicing factor SF45‐like, RNA recognition (RRM) and glycine‐rich (G‐patch) motifs. The presence of these motifs suggests that T. gondii TgDRE might also be involved inother biological functions such as RNA metabolism in addition to DNA‐repair.
Regulation of glypican‐1, syndecan‐1 and syndecan‐4 mRNAs expression by follicle‐stimulating hormone, cAMP increase and calcium influx during rat Sertoli cell development
Authors: Brucato Sylvie , Bocquet Jean , Villers Corinne
Published: March 2003

In seminiferous tubules, Sertoli cells provide structural and nutritional support for the developing germinal cells. Cell‐ to‐cell signaling and cell adhesion require proteoglycans expressed at the cell membrane. A preliminary biochemical and structural approach indicated that cell surface proteoglycans are mostly heparan sulfate proteoglycans (HSPG). Glypican‐1, syndecans‐1 and ‐4 were identified using a molecular approach. Their differential regulation was demonstrated in immature rat Sertoli cells. Follicle‐stimulating hormone (FSH) is the main regulator of Sertoli cell function. Signal transduction triggered by FSH involves both an increased intracellular cAMP synthesis and a calcium influx. This study demonstrates that FSH, through its second messengers (increase in intracellular cAMP and intracellular calcium), downregulated the glypican‐1 mRNA expression in Sertoli cells from 20‐day‐old rats. On the other hand, syndecan‐1 mRNA expression is not modulated by FSH as it would result from the antagonistic effects of increased intracellular cAMP and intracellular calcium levels. Finally, syndecan‐4 mRNA expression is not regulated by this pathway.

 The present study was extended during Sertoli cell development. Indeed, Sertoli cells undergo extensive changes during the postnatal period both in structure and function. These important transformations are critical for the establishment of spermatogenesis and development of the adult pattern of testicular function. Our data indicated that the regulation of HSPG mRNA expression is HSPG‐specific and depends on the Sertoli cell developmental stage.
Chemical structures and immunolocalization of glycosphingolipids isolated from Diphyllobothrium hottai adult worms and plerocercoids
Authors: Iriko Hideyuki , Nakamura Kazuo , Kojima Hisako , Iida‐Tanaka Naoko , Kasama Takeshi , Kawakami Yasushi , Ishizuka Ineo , Uchida Akihiko , Murata Yoshihiko , Tamai Yoichi
Published: March 2003

Glycosphingolipids (GSLs) were purified from adults and plerocercoids of the tapeworm Diphyllobothrium hottai, and their chemical structures were determined. Total lipid fractions prepared from chloroform/methanol extracts of whole tissues were fractionated successively on ion‐exchange chromatography, silicic acid column chromatography, and preparative TLC. The purified GSLs were characterized by methylation analysis, TLC‐immunostaining, liquid secondary ion MS, MALDI‐TOF MS, and 1H‐NMR. Ten GSLs were isolated from adult worms and four from plerocercoids, comprising mono‐, di‐, tri‐, tetra‐, and pentasaccharides. The GSL Galβ1–4(Fucα1–3)Glcβ1–3Galβ1‐Cer was found in adult worms but not in plerocercoids, whereas Galβ1–4 (Fucα1–3)Glcβ1–3(Galβ1–6)Galβ1‐Cer was found in both adult worms and plerocercoids. We previously found a similar series of GSLs in plerocercoids of the cestode Spirometra erinaceieuropaei, and termed them ‘spirometosides’[Kawakami, Y. et al. (1996) Eur J. Biochem. 239, 905–911]. The core structure of spirometosides, Galβ1–4Glcβ1–3 Galβ1‐Cer, may have taxonomic significance, being characteristic of pseudophyllidean tapeworms. In the present study, GSL compositions were significantly different between adults and plerocercoids, and growth‐dependent changes in composition were documented. We found a novel dihexosylceramide, Glcβ1–3Galβ1‐Cer, which is a possible precursor for spirometosides. Immunohistochemical examination showed that spirometoside GSLs are highly enriched in the inner surface of bothria, the major point of contact between the adult worm and the host's intestine. Our findings indicate that spirometosides are involved in host–parasite interaction.
NMR structure of the HIV‐1 regulatory protein Vpr in H2O/trifluoroethanol
Authors: Wecker K. , Morellet N. , Bouaziz S. , Roques B. P.
Published: March 2003

The human immunodeficiency virus type 1, HIV‐1, genome encodes a highly conserved regulatory gene product, Vpr (96 amino acids), which is incorporated into virions in quantities equivalent to those of the viral Gag protein. In infected cells, Vpr is believed to function during the early stages of HIV‐1 replication (such as transcription of the proviral genome and migration of preintegration nuclear complex), blocks cells in G2 phase and triggers apoptosis. Vpr also plays a critical role in long‐term AIDS disease by inducing viral infection in nondividing cells such as monocytes and macrophages. To gain deeper insight of the structure–function relationship of Vpr, the intact protein (residues 1–96) was synthezised. Its three‐dimensional structure was analysed using circular dichroism and two‐dimensional 1H‐ and 15N‐NMR and refined by restrained molecular dynamics. In addition, 15N relaxation parameters (T1, T2) and heteronuclear 1H‐15N NOEs were measured. The structure of the protein is characterized by a well‐defined γ turn(14–16)‐α helix(17–33)‐turn(34–36), followed by a α helix(40–48)‐loop(49–54)‐α helix(55–83) domain and ends with a very flexible C‐terminal sequence. This structural determination of the whole intact Vpr molecule provide insights into the biological role played by this protein during the virus life cycle, as such amphipathic helices are believed to be involved in protein–lipid bilayers, protein–protein and/or protein–nucleic acid interactions.
Thermodynamic stability of porcine β‐lactoglobulin
Authors: Burova Tatiana V. , Grinberg Natalia V. , Visschers Ronald W. , Grinberg Valerij Y. , de Kruif Cornelus G.
Published: March 2003

The proposed biological function of β‐lactoglobulins as transporting proteins assumes a binding ability for ligands and high stability under the acidic conditions of the stomach. This work shows that the conformational stability of nonruminant porcine β‐lactoglobulin (BLG) is not consistent with this hypothesis. Thermal denaturation of porcine BLG was studied by high‐sensitivity differential scanning calorimetry within the pH range 2.0–10.0. Dependences of the denaturation temperature and enthalpy on pH were obtained, which reveal a substantial decrease in both parameters in acidic and basic media. The denaturation enthalpy follows a linear dependence on the denaturation temperature. The slope of this line is 9.4 ± 0.6 kJ·mol−1· K−1,which is close to the denaturation heat capacity increment ΔdCp = 9.6 ± 0.5 kJ·mol−1·K−1, determined directly from the thermograms. At pH 6.25 the denaturation temperatures of porcine and bovine BLG coincide, at 83.2 °C. At this pH the denaturation enthalpy of porcine BLG is 300 kJ·mol−1. The denaturation transition of porcine BLG was shown to be reversible at pH 3.0 and pH 9.0. The transition profile at both pH values follows the two‐state model of denaturation. Based on the pH‐dependence of the transition temperature and the linear temperature dependence of the transition enthalpy, the excess free energy of denaturation, ΔdGE, of porcine BLG was calculated as a function of pH and compared with that of bovine BLG derived from previously reported data. The pH‐dependence of ΔdGE is analysed in terms of the contributions of side‐chain H‐bonds to the protein stability. Interactions stabilizing native folds of porcine and bovine BLG are discussed.
A sucrose binding protein homologue from soybean exhibits GTP‐binding activity that functions independently of sucrose transport activity
Authors: Pirovani Carlos P. , Macêdo Joci Neuby A. , Contim Luís Antônio S , Matrangolo Fabiana S. V. , Loureiro Marcelo E. , Fontes Elizabeth P. B.
Published: March 2003

The sucrose binding protein (SBP) has been implicated as an important component of the sucrose uptake system in plants. SBP‐mediated sucrose transport displays unique kinetic features and the protein is not similar to other transport proteins. Here, we report the characterization of a member of the SBP family from soybean [Glycine max (L) Merrill] designated S64 or SBP2. Subcellular fractionation and precipitation by GTP‐agarose demonstrated that S64/SBP2 is a membrane‐associated protein that exhibits GTP binding activity. Purified recombinant S64/SBP2 protein, expressed as a histidine‐tagged protein in Escherichia coli, exhibited nucleotide‐binding specificity to guanine nucleotides. The GTP binding site was mapped to an imperfect Walker A type‐sequence, Ala279‐Leu‐Ala‐Pro‐Thr‐Lys‐Lys‐Ser286, by site‐directed mutagenesis. Escherichia coli‐produced wild‐type protein and a truncated version of the protein containing the putative binding‐sequence‐bound GTP, although not with the same efficiency. In contrast, replacement of Thr283 and Lys284 residues to Leu and Glu residues prevented GTP binding. The site directed mutant failed to bind GTP but retained the ability to undergo oligomerization andto promote growth of the susy7 yeast strain, deficient inutilizing extracellular sucrose, on medium containing sucrose as the sole carbon source. Our results indicate that GTP binding and sucrose transport by SBP are separable and function independently. The implications of our findings with respect to the function and membrane topology of SBP are discussed.
Redox‐sensitive loops D and E regulate NADP(H) binding in domain III and domain I–domain III interactions in proton‐translocating Escherichia coli transhydrogenase
Authors: Johansson Carina , Pedersen Anders , Karlsson B. Göran , Rydström Jan
Published: March 2003

Membrane‐bound transhydrogenases are conformationally driven proton‐pumps which couple an inward proton translocation to the reversible reduction of NADP+ by NADH (forward reaction). This reaction is stimulated by an electrochemical proton gradient, Δp, presumably through an increased release of NADPH. The enzymes have three domains: domain II spans the membrane, while domain I and III are hydrophilic and contain the binding sites for NAD(H) and NADP(H), respectively. Separately expressed domain I and III together catalyze a very slow forward reaction due to tightly bound NADP(H) in domain III.

With the aim of examining the mechanistic role(s) of loop D and E in domain III and intact cysteine‐free Escherichia coli transhydrogenase by cysteine mutagenesis, the conserved residues βA398, βS404, βI406, βG408, βM409 and βV411 in loop D, and residue βY431 in loop E were selected. In addition, the previously made mutants βD392C and βT393C in loop D, and βG430C and βA432C in loop E, were included. All loop D and E mutants, especially βI406C and βG430C, showed increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild‐type enzyme. Determination of values indicated that the former increase was due to a strongly increased dissociation of NADPH caused by an altered conformation of loops D and E. In contrast, the cysteine‐free G430C mutant of the intact enzyme showed the same inhibition of both forward and reverse rates. Most domain III mutants also showed a decreased affinity for domain I. The results support an important and regulatory role of loops D and E in the binding of NADP(H) as well as in the interaction between domain I and domain III.
Proximal ligand motions in H93G myoglobin
Authors: Franzen Stefan , Peterson Eric S. , Brown Derek , Friedman Joel M , Thomas Melissa R. , Boxer Steven G.
Published: March 2003

Resonance Raman spectroscopy has been used to observe changes in the iron–ligand stretching frequency in photoproduct spectra of the proximal cavity mutant of myoglobin H93G. The measurements compare the deoxy ferrous state of the heme iron in H93G(L), where L is an exogenous imidazole ligand bound in the proximal cavity, to the photolyzed intermediate of H93G(L)*CO at 8 ns. There are significant differences in the frequencies of the iron–ligand axial out‐of‐plane mode ν(Fe–L) in the photoproduct spectra depending on the nature of L for a series of methyl‐substituted imidazoles. Further comparison was made with the proximal cavity mutant of myoglobin in the absence of exogenous ligand (H93G) and the photoproduct of the carbonmonoxy adduct of H93G (H93G‐*CO). For this case, it has been shown that H2O is the axial (fifth) ligand to the heme iron in the deoxy form of H93G. The photoproduct of H93G‐*CO is consistent with a transiently bound ligand proposed to be a histidine. The data presented here further substantiate the conclusion that a conformationally driven ligand switch exists in photolyzed H93G‐*CO. The results suggest that ligand conformational changes in response to dynamic motions of the globin on the nanosecond and longer time scales are a general feature of the H93G proximal cavity mutant.