bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of ENSO on East African ecosystems: a multivariate analysis based on climate and remote sensing data
Authors: Plisnier P. D. , Serneels S. , Lambin E. F.
Published: March 2003

1 The El Niño‐Southern Oscillation (ENSO) is an important driver of inter‐annual variations in climate and ecosystem productivity in tropical regions. Most previous studies have analysed ENSO‐induced changes in climate based on a single variable, such as rainfall. Also, it is generally assumed that the ENSO impact in East Africa is geographically uniform.

2  The objective of this study is to improve understanding of the impact of ENSO on East African ecosystems, by measuring teleconnections between an ENSO index and a number of ecosystem variables in a spatially explicit way and for different time lags. We analysed the spatial patterns of teleconnections in the region by combining time series of climate variables measured for meteorological stations with time series of a vegetation index and surface temperature data measured by remote sensing.

3 Our results confirm the ENSO impact on the climatic and ecological variability in East Africa. However, the pattern of teleconnections is much more complex than generally assumed, both in terms of spatial distribution and impact on different ecosystem variables. Not all climate and land surface variables are teleconnected to ENSO in the same way, which leads to a complex impact of ENSO on the ecosystem. Moreover, the ENSO impact is highly differentiated in space, as the direction, magnitude and timing of this impact are controlled by the local climate system, the presence of large lakes, proximity to the coast and, possibly, local topography and land cover.
Functional roles of remnant plant populations in communities and ecosystems
Authors: Eriksson Ove
Published: March 2003

A hypothesis is suggested for functional roles of remnant plant populations in communities and ecosystems. A remnant population is capable of persistence during extended time periods, despite a negative population growth rate, due to long‐lived life stages and life‐cycles, including loops that allow population persistence without completion of the whole life cycle. A list of critera is suggested to help identification of remnant plant populations. Several community and ecosystem features may result from the presence of remnant plant populations. Apart from increasing community and ecosystem resilience just by being present, remnant populations may contribute to resilience through enhancing colonization by other plant species, by providing a persistent habitat for assemblages of animals and microorganisms, and by reducing variation in nutrient cycling. It is suggested that the common ability of plants to develop remnant populations is a contributing factor to ecosystem stability. Remnant populations are important for the capacity of ecosystems to cope with the present‐day impact caused by human society, and their occurrence should be recognized in surveys of threatened plant species and communities.
Matrix metalloproteinase‐2 from bronchial epithelial cells induces the proliferation of subepithelial fibroblasts
Authors: Xu J. , Benyon R. C. , Leir S.‐H. , Zhang S. , Holgate S. T. , Lackie P. M.
Published: March 2003

Background In bronchial asthma, subepithelial fibrosis in the conducting airways is associated with increased numbers of subepithelial fibroblasts.

Objective This study examined the hypothesis that MMP‐2 from airway epithelial cells induces the proliferation of subepithelial fibroblasts.

Methods Using primary bronchial epithelial cells MMP‐2, MT1‐MMP and TIMP‐2 mRNA expression were assessed by Northern blotting and RT‐PCR. Primary bronchial epithelial cells transfected with constructs encoding pro‐MMP‐2 and MT1‐MMP (MMP‐14).

Results Transfected cells showed enhanced expression of the appropriate mRNA species by RT‐PCR and enhanced MMP‐2 or MT1‐MMP activity by zymography. Active MMP‐2 levels in epithelial supernatants were increased most by cotransfection with pro‐MMP‐2 and MT1‐MMP encoding constructs. By measuring tritiated thymidine incorporation, supernatants from transfected cells were found to enhance DNA synthesis of primary airway fibroblast cultures compared with controls. There was a strong correlation (r = 0.9, P < 0.01) between MMP‐2 levels in epithelial cell conditioned media and fibroblast proliferation as indicated by DNA synthesis. The MMP inhibitor 1,10‐phenanthroline attenuated the increased proliferation, while the addition of exogenous purified MMP‐2 alone also increased fibroblast proliferation.

Conclusions Our results support a role for MMP‐2 in mediating cross‐talk between epithelial cells and myofibroblasts.
Dexamethasone inhibits maturation, cytokine production and FcεRI expression of human cord blood‐derived mast cells
Authors: Smith S. J. , Piliponsky A. M. , Rosenhead F. , Elchalal U. , Nagler A. , Levi‐Schaffer F.
Published: March 2003

Background Mast cells are responsible for eliciting the early phase and for contributing to the development of the late phase of allergic reactions, through the release of cytokines and other inflammatory mediators.

Objective To assess whether the glucocorticoid dexamethasone has a direct effect on mast cell progenitor maturation and on mature cord blood‐derived mast cell properties.

Methods Mast cells were obtained by culturing human umbilical cord blood mononuclear cells with stem cell factor, IL‐6 and prostaglandin E2. Mast cell numbers were assessed by Toluidine Blue staining and immunocytochemistry of tryptase positive cells. The expression of FcεRI, CD49d and c‐kit was assessed by flow cytometry. Histamine release was determined by a radioenzymatic assay. Cys‐LT, GM‐CSF and TNF‐α production and release were determined by ELISA.

Results Dexamethasone (10−6 M−10−9 M) time‐ and dose‐dependently inhibited the maturation of the mast cell progenitors. Dexamethasone did not affect the basal expression of FcεRI, CD49d and c‐kit, but it inhibited the IgE‐dependent enhanced expression of FcεRI. Dexamethasone (10−6 M−10−9 M) had no significant effect on FcεRI‐dependent histamine release or the synthesis and release of Cys‐LT from the mature mast cells. However, pre‐incubation of the mast cell cultures with dexamethasone for 1 h, prior to cross‐linking of FcεRI, dose‐dependently inhibited the production and secretion of both GM‐CSF and TNF‐α.

Conclusions From these in vitro data we propose that glucocorticosteroids are effective drugs in the management of allergic inflammation due to their capacity to inhibit mast cell development, IgE‐dependent FcεRI expression and mast cell production of GM‐CSF and TNF‐α.
Effects of fexofenadine and desloratadine on subjective and objective measures of nasal congestion in seasonal allergic rhinitis
Authors: Wilson A. M. , Haggart K. , Sims E. J. , Lipworth B. J.
Published: March 2003

Background In vitro
studies have shown much higher H
1 ‐receptor antagonist potency with desloratadine (DL) compared to fexofenadine (FEX), although it is unclear whether this has any clinical relevance on disease control parameters in seasonal allergic rhinitis (SAR), especially for nasal congestion.

Objective To compare the relative efficacy between presently recommended doses of DL and FEX on daily measurements of peak nasal inspiratory flow (PNIF) and nasal symptoms in SAR.

Methods Forty‐nine patients with SAR were randomized into a double‐blind, placebo‐controlled cross‐over study during the grass pollen season, comparing 2 weeks of once daily treatment with (a) 180 mg FEX or (b) 5 mg DL, taken in the morning. There was a 7–10 day placebo run‐in and washout prior to each randomized treatment. Measurements were made in the morning (am) and in the evening (pm) for PNIF (the primary outcome variable), nasal and eye symptoms. The average of am/pm values were used for analysis.

Results There were significant (P < 0.05) improvements, compared to placebo, with FEX and DL, for PNIF, nasal blockage, nasal irritation, and total nasal symptoms, but not nasal discharge or eye symptoms. There were no significant differences between active treatments. Values for PNIF (L/min) for mean placebo baseline, mean difference from baseline (95% CI for difference) were 126, 10 (4–16) for FEX; and 122, 11 (4–17) for DL. The mean difference (95% CI) between FEX vs. DL was 1 L/min (−7–8). Values for total nasal symptoms (out of 12) were: 3.2, 0.7 (0.2–1.2) for FEX; and 3.4, 0.9 (0.3–1.5) for DL, and for nasal blockage (out of 3) were: 1.1, 0.2 (0.1–0.4) for FEX; and 1.2, 0.3 (0.1–0.5) for DL. The mean difference (95% CI) in total nasal symptoms and nasal blockage between FEX vs. DL was 0.1 (−0.6–0.8) and 0.1 (−0.2–0.3), respectively.

Conclusions Recommended once daily doses of fexofenadine and desloratadine were equally effective in improving nasal peak flow and nasal symptoms in SAR.
Past and future of predictions in plant invasions: a field test by time
Authors: Pyšek Petr
Published: March 2003

Intensive interest in the alien flora of the Czech Republic stimulated one of the earliest attempts (in the early 1970s) to predict potential invaders of arable land. Another paper published recently by the same research team using the same methodology provides a relatively unique possibility to assess the success of these predictions after a quarter of a century. The predictions were successful for 39.3% of the 28 species included, while 60.7% of invasion cases must be considered as failures. Prediction was rather unsuccessful for the members of Asteraceae (14.3%), and more correct for annuals/biennials (45.5%) than for perennials (16.7%). No pattern was found with respect to the area of origin. The results indicate that past predictions based largely on intuition were less successful than modern prediction systems using the knowledge of a large number of characters and carried out using advanced computation methods. The correct identification of invaders using such systems reaches values between 61% and 91%.
The role of doctors, patients and managers in priority setting decisions: lessons from the `Child B' case
Authors: Ham Chris
Published: March 2003

The difficulties in treating patients with life threatening illnesses were highlighted by the case of Jaymee Bowen, a 10‐year‐old girl with leukaemia who was refused funding for a second bone marrow transplant in 1995. Jaymee's case was widely reported at the time and came to epitomize the dilemmas of rationing in the United Kingdom's National Health Service. In reality, the paediatricians who had cared for Jaymee based their decision on clinical rather than financial considerations, and the media reporting of the case failed to reflect the complexities of the issues involved. The case also demonstrated the difficulties of determining the best interests of children and of obtaining their consent to treatment. There were disagreements between Jaymee's father and the paediatricians who had treated her about how her best interests could be served and this led to a breakdown of trust and the search for further opinions. This highlighted the rise of consumerism in health care and the challenge to doctors and managers to justify their decisions and to give reasons for these decisions. The common theme in Jaymee's story is the need for greater openness in decisions on priority setting and stronger safeguards for patients.
Patient participation groups in general practice in the National Health Service
Authors: Brown Ian
Published: March 2003

General practice remains the organizational hub of first level health services in the United Kingdom. Patient participation groups are probably the most well known model for public participation in this setting and, although still not widespread, they have been a slowly expanding area of development for almost three decades. This paper sets out to critically asses patient participation groups in general practice by considering the context of their development and reviewing the research literature about groups. Critical issues needing more study and key methodological challenges are then discussed.

Patient participation groups have been a somewhat shifting and contested phenomenon, embracing trends and changing as policy priorities have changed over the years. There is some evidence to think that they might have potential as a local element within a public participation strategy in the National Health Service. However, the field studies are very limited and more research of a better quality is needed. The state of knowledge is not adequate to be able to say with any confidence if or how such groups should be developed. A better understanding is needed of the public’s perspectives on this and other models of participation. There are many other questions to do with patient participation groups’ purpose, equitable access, and effectiveness that need to be addressed. The methodological challenges include issues of how to involve all stakeholders in the research process; and how to study less tangible aspects of general practice organization, such as culture and power, that effect the public’s participation.
Involving patients in decisions regarding preventive health interventions using the analytic hierarchy process
Authors: Dolan James G.
Published: March 2003

Practice guidelines that recommend active patient involvement in decisions about preventive health interventions are becoming increasingly common. These decisions frequently involve difficult trade‐offs between competing risks and benefits that require easily accessible information about the expected outcomes, superb doctor–patient communication, and effective integration of objective outcome data with individual values and preferences. Successful implementation of recommendations for shared decision‐making in preventive health care will require the development of efficient methods for making these complex decisions in busy practice settings. This article describes how the analytic hierarchy process, a multiple criteria decision‐making method, could facilitate successful implementation of shared decision‐making regarding preventive health care in clinical practice. The method is illustrated using recent guidelines for colorectal cancer screening for average risk patients issued by the American Gastroenterological Association.
Public perceptions about low back pain and its management: a gap between expectations and reality?
Authors: Moffett Jennifer A. Klaber , Newbronner Elizabeth , Waddell Gordon , Croucher Karen , Spear Steven
Published: March 2003

Objective

To compare public perceptions and patient perceptions about back pain and its management with current clinical guidelines.

Design

A survey using a quota sampling technique.

Setting

On‐the‐street in South Derbyshire in the UK.

Subjects

507 members of the general population aged between 20 and 60 years, including a representative subsample of 40% who had experienced back pain in the previous year.

Survey

To test knowledge and perceptions of back pain and its best management using statements based on The Back Book which was produced in conjunction with the Royal College of General Practitioners and based on best available evidence. In addition expectations of back pain management and outcome were investigated.

Results

Forty percent of this sample had experienced back pain during the previous year, more than half of whom had consulted their GP. More than half believed the spine is one of the strongest part of the body, but nearly two thirds incorrectly believed that back pain is often due to a slipped disc or trapped nerve. Two thirds expected a GP to be able to tell them exactly what was wrong with their back, although slightly fewer among those who had consulted. Most expected to have an X‐ray, especially if they had consulted. Most recognised that the most important thing a GP can do is offer reassurance and advice. The responses were not related to age, gender or social class. Those who had consulted appeared to have slightly more misconceptions: this could be partly due to people with more severe problems or more misconceptions being more likely to consult, but also suggests either that GPs are still giving inaccurate information or at least failing to correct these misconceptions.

Conclusions

The problem of managing back pain might be reduced by closing the gap between the public’s expectations and what is recommended in the guidelines through the promotion of appropriate health education messages. Further professional education of GPs also appears to be needed to update them in the most effective approach to managing back pain.
Challenges of participatory research: reflections on a study with breast cancer self‐help groups
Authors: Gray Ross E. , Fitch Margaret , Davis Christine , Phillips Catherine
Published: March 2003

Objective

To review and discuss issues related to participatory research, as they apply within the arena of cancer control.

Design

A participatory research study with breast cancer self‐help groups is referred to for description and discussion purposes. That study employed primarily individual and group interviews to assess benefits and limitations of self‐help groups.

Settings

Four breast cancer self‐help groups in Ontario communities provided the core involvement in the participatory research project.

Results

The values and practices of mainstream academic research often conflict with those of research emphasizing participation and control of communities under study, leading to a variety of challenges for the latter approaches. Practical constraints faced by many community groups have important implications for participatory research approaches.

Conclusions

A balance needs to be found for participatory research within cancer control – one that ensures that the core aims of participatory research are maintained, while simultaneously acknowledging the various challenges that make a fully participatory project unrealistic. Steps can be taken to achieve a workable balance.
Consulting about consulting: challenges to effective consulting about public health research
Authors: Graham Janne , Broom Dorothy , Whittaker Andrea
Published: March 2003

Objective To understand barriers to obtaining input from consumers in developing public health research.

Methods Documentation of a failed attempt at consumer consultation supplies information on barriers to effective involvement and conditions that must prevail to improve consultation.

Results People are keen to be heard in the formulation of health research. However, competing demands and limited resources make it difficult for community groups to allocate scarce resources to consultation. Sometimes research issues may seem ‘academic’ and thus remote from the urgent priorities of the people with whom researchers wish to consult. Consultation may require more time than researchers on limited budgets can afford.

Conclusions Despite a general public health commitment to involving consumers in research development, obstacles to consultation make it difficult to incorporate it into the research agenda.

Implications Researchers and funding bodies will need to allocate resources to consumer consultation if it is to become the rule rather than the exception in public health research.
The ethics of evidence‐based patient choice
Authors: Parker Michael
Published: March 2003

In this paper I analyse the ethical implications of the concept of ‘evidence‐based patient choice’ in the light of criticism of the ‘individualism’ of patient‐centred medicine. I argue that individualism in the sense used by the critics of patient centred medicine is not an inevitable consequence of an emphasis on patient choice and that a concern with the promotion of individual choices is not incompatible with ‘communitarian’ values.

Indeed, I argue that any ethical approach to decision‐making in health‐care must be capable of taking seriously both the moral status of the individual (and of his or her choices) and the moral significance of the social dimensions of such choices. The best way to ensure respect for the principle of autonomy, I suggest, is to facilitate and encourage social interactions of a particular, deliberative, kind. This is also the best way to ensure that the broader public interest is taken into account in decision‐making.
A decision aid for men with early stage prostate cancer: theoretical basis and a test by surrogate patients
Authors: Feldman‐Stewart Deb , Brundage Michael D. , Van Manen Lori
Published: March 2003

Background We developed a decision aid for patients with curable prostate cancer based on Svenson’s DiffCon Theory of Decision Making. This study was designed to determine if surrogate patients using the aid could understand the information presented, complete all tasks, show evidence of differentiation, and arrive at a preferred treatment choice.

Methods Men, at least 50 years old and never diagnosed with prostate cancer, were recruited through local advertisements. Participants were asked to imagine that they were a case‐scenario patient. Then they completed the decision aid interview, which included three components: (i) information presentation, with comprehension questions, (ii) exercises to help identify attributes important to the decision, and (iii) value‐clarification exercises.

Results Sixty‐nine men volunteered. They had a mean age of 61.2 (range 50–83) years, 37% had no formal education beyond high school, and 87% were living with a partner. All participants completed all aspects of the interview. They answered an average of 10 comprehension questions each, with a mean of 94.7% correct without a prompt. Each attribute in the information presented was identified by at least one participant as important to his decision. Participants identified a median of five attributes as important (ranges 1–14) at each of three points during the interview; 75% changed at least one important attribute during the interview. Forty‐nine per cent of participants also identified attributes as important that were not included in the presented information. Participants showed a wide range of values in each of seven trade‐off exercises. Eighty‐eight per cent of participants showed evidence of differentiation; 75% had a clear treatment preference by the end of the interview.

Conclusions Our decision aid appears to meet its goals for surrogate patients and illustrates the strengths of the DiffCon theory. The ability of the aid to accommodate wide variability, both in information needs and in important attributes, is a particular strength of the decision aid. It now requires testing in patients with prostate cancer.
Purification and characterization of the thyrotropin‐releasing hormone (TRH)‐degrading serum enzyme and its identification as a product of liver origin
Authors: Schmitmeier Stephanie , Thole Hubert , Bader Augustinus , Bauer Karl
Published: March 2003

Previous biochemical studies have indicated that the membrane‐bound thyrotropin‐releasing hormone (TRH)‐degrading enzyme (TRH‐DE) from brain and liver and the serum TRH‐DE are derived from the same gene. These studies also suggested that the serum enzyme is of liver origin. The present study was undertaken to verify these hypotheses. In different species, a close relationship between the activities of the serum enzyme and the particulate liver enzyme was noticed. The activity of the serum enzyme decreased when rats were treated with thioacetamide, a known hepatotoxin. With hepatocytes cultured in a sandwich configuration, release of the TRH‐DE into the culture medium could also be demonstrated.

The trypsin‐solubilized particulate liver TRH‐DE and the serum TRH‐DE were purified to electrophoretic homogeneity. Both enzymes and the brain TRH‐DE were recognized by a monoclonal antibody generated with the purified brain enzyme as antigen. Lectin blot analysis indicated that the serum enzyme and the liver enzyme are glycoproteins containing a sugar structure of the complex type, whereas the brain enzyme exhibits an oligomannose/hybrid glycostructure. A molecular mass of 97 000 Da could be estimated for all three enzymes after deglycosylation and SDS/PAGE followed by Western blotting.

Fragment analysis of the serum TRH‐DE revealed that the peptide sequences correspond to the cDNA deduced amino‐acid sequences of the membrane‐bound brain TRH‐DE, whereby two peptides were identified that are encoded by exon 1. These data strongly support the hypothesis that the TRH‐DEs are all derived from the same gene, whereby the serum enzyme is generated by proteolytic cleavage of the particulate liver enzyme.
Functional epitope of common γ chain for interleukin‐4 binding
Authors: Zhang Jin‐Li , Buehner Manfred , Sebald Walter
Published: March 2003

Interleukin 4 (IL‐4) can act on target cells through an IL‐4 receptor complex consisting of the IL‐4 receptor α chain and the common γ chain (γc). An IL‐4 epitope for γc binding has previously been identified. In this study, the γc residues involved in IL‐4 binding were defined by alanine‐scanning mutational analysis. The epitope comprises γc residues I100, L102, and Y103 on loop EF1 together with L208 on loop FG2 as the major binding determinants. These predominantly hydrophobic determinants interact with the hydrophobic IL‐4 epitope composed of residues I11, N15, and Y124. Double‐mutant cycle analysis revealed co‐operative interaction between γc and IL‐4 side chains. Several γc residues involved in IL‐4 binding have been previously shown to be mutated in X‐linked severe combined immunodeficiency. The importance of these binding residues for γc function is discussed. These results provide a basis for elucidating the molecular recognition mechanism in the IL‐4 receptor system and a paradigm for other γc‐dependent cytokine receptor systems.
The opgGIH and opgC genes of Rhodobacter sphaeroides form an operon that controls backbone synthesis and succinylation of osmoregulated periplasmic glucans
Authors: Cogez Virginie , Gak Evgueni , Puskas Agnes , Kaplan Samuel , Bohin Jean‐Pierre
Published: March 2003

Osmoregulated periplasmic glucans (OPGs) of Rhodobacter sphaeroides are anionic cyclic molecules that accumulate in large amounts in the periplasmic space in response to low osmolarity of the medium. Their anionic character is provided by the substitution of the glucosidic backbone by succinyl residues. A wild‐type strain was subject to transposon mutagenesis, and putative mutant clones were screened for changes in OPGs by thin layer chromatography. One mutant deficient in succinyl substitution of the OPGs was obtained and the gene inactivated in this mutant was characterized and named opgC. opgC is located downstream of three ORFs, opgGIH, two of which are similar to the Escherichia coli operon, mdoGH, governing OPG backbone synthesis. Inactivation of opgG, opgI or opgH abolished OPG production and complementation analysis indicated that the three genes are necessary for backbone synthesis. In contrast, inactivation of a gene similar to ndvB, encoding the OPG‐glucosyl transferase in Sinorhizobium meliloti, had no consequence on OPG synthesis in Rhodobacter sphaeroides. Cassette insertions in opgH had a polar effect on glucan substitution, indicating that opgC is in the same transcription unit. Expression of opgIHC in E. coli mdoB/mdoC and mdoH mutants allowed the production of slightly anionic and abnormally long linear glucans.
Structural determination of lipid A of the lipopolysaccharide from Pseudomonas reactans
Authors: Silipo Alba , Lanzetta Rosa , Garozzo Domenico , Lo Cantore Pietro , Iacobellis Nicola Sante , Molinaro Antonio , Parrilli Michelangelo , Evidente Antonio
Published: March 2003

The chemical structure of lipid A from the lipopolysaccharide of the mushroom‐associated bacterium Pseudomonas reactans, a pathogen of cultivated mushroom, was elucidated by compositional analysis and spectroscopic methods (MALDI‐TOF and two‐dimensional NMR). The sugar backbone was composed of the β‐(1′→6)‐linked d‐glucosamine disaccharide 1‐phosphate. The lipid A fraction showed remarkable heterogeneity with respect to the fatty acid and phosphate composition. The major species are hexacylated and pentacylated lipid A, bearing the (R)‐3‐hydroxydodecanoic acid [C12:0 (3OH)] in amide linkage and a (R)‐3‐hydroxydecanoic [C10:0 (3OH)] in ester linkage while the secondary fatty acids are present as C12:0 and/or C12:0 (2‐OH). A nonstoichiometric phosphate substitution at position C‐4′ of the distal 2‐deoxy‐2‐amino‐glucose was detected. Interestingly, the pentacyl lipid A is lacking a primary fatty acid, namely the C10:0 (3‐OH) at position C‐3′. The potential biological meaning of this peculiar lipid A is also discussed.
Identification of ectopic anionic trypsin I in rat lungs potentiating pneumotropic virus infectivity and increased enzyme level after virus infection
Authors: Towatari Takae , Ide Mikiko , Ohba Kumiko , Chiba Yuusuke , Murakami Meiko , Shiota Mayumi , Kawachi Miki , Yamada Hiroshi , Kido Hiroshi
Published: March 2003

Extracellular cleavage of virus envelope fusion glycoproteins by host cellular proteases is a prerequisite for the infectivity of mammalian and nonpathogenic avian influenza viruses, and Sendai virus. In search of such target processing proteases in the airway, we recently found a new candidate trypsin‐like processing protease in rat lungs, which was induced by Sendai virus infection, and identified as ectopic rat anionic trypsin I. On SDS/PAGE under reducing and nonreducing conditions, the purified enzyme gave protein bands corresponding to 29 and 22 kDa, respectively, i.e. at the same positions as rat pancreatic anionic trypsin I. It exhibited an apparent molecular mass of 31 kDa on molecular sieve chromatography and its isoelectric point was pH 4.7. The amino‐acid sequences of the N‐terminus and proteolytic digest peptides of the purified enzyme were consistent with those of rat pancreatic anionic trypsin I. Its substrate specificities and inhibitor sensitivities were the same as those of the pancreatic enzyme. The purified enzyme efficiently processed the fusion glycoprotein precursor of Sendai virus and hemagglutinin of human influenza A virus, and potentiated the infectivity of Sendai virus in the same dose‐dependent manner as the pancreatic one. Immunohistochemical studies revealed that this protease is located in the stromal cells in peri‐bronchiolar regions. These results suggest that ectopic anionic trypsin I in rat lungs induced by virus infection may trigger virus spread in rat lungs.
Functional expression and mutational analysis of flavonol synthase from Citrus unshiu
Authors: Wellmann Frank , Lukačin Richard , Moriguchi Takaya , Britsch Lothar , Schiltz Emile , Matern Ulrich
Published: March 2003

Flavonols are produced by the desaturation of flavanols catalyzed by flavonol synthase. The enzyme belongs to the class of intermolecular dioxygenases which depend on molecular oxygen and FeII/2‐oxoglutarate for activity, and have been in focus of structural studies recently. Flavonol synthase cDNAs were cloned from six plant species, but none of the enzymes had been studied in detail. Therefore, a cDNA from Citrus unshiu (Satsuma mandarin) designated as flavonol synthase was expressed in Escherichia coli, and the purified recombinant enzyme was subjected to kinetic and mutational chacterizations. The integrity of the recombinant synthase was revealed by a molecular ion from MALDI‐TOF mass spectrometry at m/z 37888 ± 40 (as compared to 37899 Da calculated for the translated polypeptide), and by partial N‐terminal sequencing. Maximal flavonol synthase activity was observed in the range of pH 5–6 with dihydroquercetin as substrate and a temperature optimum at about 37 °C. Km values of 272, 11 and 36 µm were determined for dihydroquercetin, FeII and 2‐oxoglutarate, respectively, with a sixfold higher affinity to dihydrokaempferol (Km 45 µm). Flavonol synthase polypeptides share an overall sequence similarity of 85% (47% identity), whereas only 30–60% similarity were apparent with other dioxygenases. Like the other dioxygenases of this class, Citrus flavonol synthase cDNA encodes eight strictly conserved amino‐acid residues which include two histidines (His221, His277) and one acidic amino acid (Asp223) residue for FeII‐coordination, an arginine (Arg287) proposed to bind 2‐oxoglutarate, and four amino acids (Gly68, His75, Gly261, Pro207) with no obvious functionality. Replacements of Gly68 and Gly261 by alanine reduced the catalytic activity by 95%, while the exchange of these Gly residues for proline completely abolished the enzyme activity. Alternatively, the substitution of Pro207 by glycine hardly affected the activity. The data suggest that Gly68 and Gly261, at least, are required for proper folding of the flavonol synthase polypeptide.
Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase
Authors: Oliveira Vitor , Gatti Reynaldo , Rioli Vanessa , Ferro Emer S. , Spisni Alberto , Camargo Antonio C. M. , Juliano Maria A. , Juliano Luiz
Published: March 2003

We report the recombinant neurolysin and thimet oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz‐GGFLRRXQ‐EDDnp (Abz, ortho‐aminobenzoicacid; EDDnp, N‐(2,4‐dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R–R or at R–X bonds, and TOP hydrolyzed at R–R or L–R bonds, showing a preference to cleave at three or four amino acids from the C‐terminal end. The kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. The relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.05–0.2 m for NaCl, KCl, NH4Cl and NaI, and 0.025–0.1 m for Na2SO4. Concentration higher than 0.2 N NH4Cl andNaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na2SO4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na2SO4, while NH4Cl and NaI have very modest effect. High positive values of enthalpy (ΔH*) and entropy (ΔS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. The effects of low temperature and high NaCl concentration on the hydrolytic activities of neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far‐UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.
Characterization and functional expression of cDNAs encoding thyrotropin‐releasing hormone receptor from Xenopus laevis
Authors: Bidaud Isabelle , Lory Philippe , Nicolas Pierre , Bulant Marc , Ladram Ali
Published: March 2003

Thyrotropin‐releasing hormone receptor (TRHR) has already been cloned in mammals wherethyrotropin‐releasing hormone (TRH) is known to act as a powerful stimulator of thyroid‐stimulating hormone (TSH) secretion. The TRH receptor of amphibians has not yet been characterized, although TRH is specifically important in the adaptation of skin color to environmental changes via the secretion of α‐melanocyte‐stimulating hormone (α‐MSH). Using a dege‐nerate PCR strategy, we report on the isolation of three distinct cDNA species encoding TRHR from the brain of Xenopus laevis. We have designated these as xTRHR1, xTRHR2 and xTRHR3. Analysis of the predicted amino acid sequences revealed that the three Xenopus TRHRs are only 54–62% identical and contain all the highly conserved residues constituting the TRH binding pocket. Amino acid sequences and phylogenetic analysis revealed that xTRHR1 is a member of TRHR subfamily 1 and xTRHR2 belongs to subfamily 2, while xTRHR3 is a new TRHR subtype awaiting discovery in other animal species. The three Xeno‐pus TRHRs have distinct patterns of expression. xTRHR3 was abundant in the brain and much scarcer in the peripheral tissues, whereas xTRHR1 was found mainly in the stomach and xTRHR2 in the heart. The Xenopus TRHR subtype 1 was found specifically in the intestine, lung and urinary bladder. These observations suggest that the three xTRHRs each have specific functions that remain to be elucidated. Expression in Xenopus oocytes and HEK‐293 cells indicates that the three Xenopus TRHRs are fully functional and are coupled to the inositol phosphate/calcium pathway. Interestingly, activation of xTRHR3 required larger concentrations of TRH compared with the other two receptors, suggesting marked differences in receptor binding, coupling or regulation.
Identification and characterization of a mammalian 14‐kDa phosphohistidine phosphatase
Authors: Ek Pia , Pettersson Gunilla , Ek Bo , Gong Feng , Li Jin‐Ping , Zetterqvist Örjan
Published: March 2003

Protein histidine phosphorylation in eukaryotes has been sparsely studied compared to protein serine/threonine and tyrosine phosphorylation. In an attempt to rectify this by probing porcine liver cytosol with the phosphohistidine‐containing peptide succinyl‐Ala‐His(P)‐Pro‐Phe‐p‐nitroanilide (phosphopeptide I), we observed a phosphatase activity that was insensitive towards okadaic acid and EDTA. This suggested the existence of a phosphohistidine phosphatase different from protein phosphatase 1, 2A and 2C. A 1000‐fold purification to apparent homogeneity gave a 14‐kDa phosphatase with a specific activity of 3 µmol·min−1·mg−1 at pH 7.5 with 7 µm phosphopeptide I as substrate. Partial amino‐acid sequence determination of the purified porcine enzyme by MS revealed similarity with a human sequence representing a human chromosome 9 gene of hitherto unknown function. Molecular cloning from a human embryonic kidney cell cDNA‐library followed by expression and purification, yielded a protein with a molecular mass of 13 700 Da, and an EDTA‐insensitive phosphohistidine phosphatase activity of 9 µmol·min−1·mg−1 towards phosphopeptide I. No detectable activity was obtained towards a set of phosphoserine‐, phosphothreonine‐, and phosphotyrosine peptides. Northern blot analysis indicated that the human phosphohistidine phosphatase mRNA was present preferentially in heart and skeletal muscle. These results provide a new tool for studying eukaryotic histidine phosphorylation/dephosphorylation.
Modulation of the oligomeric structures of HIV‐1 retroviral enzymes by synthetic peptides and small molecules
Authors: Sluis‐Cremer Nicolas , Tachedjian Gilda
Published: March 2003

The efficacy of antiretroviral agents approved for the treatment of HIV‐1 infection is limited by the virus's ability to develop resistance. As such there is an urgent need for new ways of thinking about anti‐HIV drug development, and accordingly novel viral and cellular targets critical to HIV‐1 replication need to be explored and exploited. The retroviral RNA genome encodes for three enzymes essential for viral replication: HIV‐1 protease (PR), HIV‐1 reverse transcriptase (RT) and HIV‐1 integrase (IN). The enzymatic functioning of each of these enzymes is entirely dependent on their oligomeric structures, suggesting that inhibition of subunit‐subunit assembly or modulation of their quaternary structures provide alternative targets for HIV‐1 inhibition. This review discusses the recent advances in the design and/or identification of synthetic peptides and small molecules that specifically target the subunit–subunit interfaces of these retroviral enzymes, resulting in the inactivation of their enzymatic functioning.
DNA and RNA damage by Cu(II)‐amikacin complex
Authors: Jeżowska‐Bojczuk Małgorzata , Szczepanik Wojciech , Leśniak Wojciech , Ciesiołka Jerzy , Wrzesiński Jan , Bal Wojciech
Published: March 2003

The oxidation‐promoting reactivity of copper(II) complex of aminoglycosidic antibiotic amikacin [Cu(II)‐Ami] in the presence of hydrogen peroxide, was studied at pH 7.4, using 2′‐deoxyguanosine (dG), pBR322 plasmid DNA and yeast tRNAPhe as target molecules. The mixtures of complex with H2O2 were found to be efficient oxidants, converting dG to its 8‐oxo derivative, generating strand breaks in plasmid DNA and multiple cleavages in tRNAPhe. The complex underwent autooxidation as well, with amikacin hydroperoxides as likely major products. This reactivity pattern was found to be due to a combination of metal‐bound and free hydroxyl radicals.
The refolding of type II shikimate kinase fromErwinia chrysanthemiafter denaturation in urea
Authors: Cerasoli Eleonora , Kelly Sharon M. , Coggins John R. , Boam Deborah J. , Clarke David T. , Price Nicholas C.
Published: March 2003

Shikimate kinase was chosen as a convenient representative example of the subclass of α/β proteins with which to examine the mechanism of protein folding. In this paper we report on the refolding of the enzyme after denaturation in urea. As shown by the changes in secondary and tertiary structure monitored by far UV circular dichroism (CD) and fluorescence, respectively, the enzyme was fully unfolded in 4 murea. From an analysis of the unfolding curve in terms of the two‐state model, the stability of the folded state could be estimated as 17 kJ·mol−1. Approximately 95% of the enzyme activity could be recovered on dilution of the urea from 4 to 0.36 m. The results of spectroscopic studies indicated that refolding occurred in at least four kinetic phases, the slowest of which (k = 0.009 s−1) corresponded with the regain of shikimate binding and of enzyme activity. The two most rapid phases were associated with a substantial increase in the binding of 8‐anilino‐1‐naphthalenesulfonic acid with only modest changes in the far UV CD, indicating that a collapsed intermediate with only partial native secondary structure was formed rapidly. The relevance of the results to the folding of other α/β domain proteins is discussed.
Secretion of pigment epithelium‐derived factor
Authors: Shao Hanshuang , Schvartz Iris , Shaltiel Shmuel
Published: March 2003

Pigment epithelium‐derived factor (PEDF), a neurotrophic and antiangiogenic protein, is an extracellular component of the retinal interphotoreceptor matrix which has been shown to be secreted by human fetal retinal pigment epithelial cells. It belongs to the serpin superfamily and contains the typical exposed reactive center loop. The function of this loop is still unknown. In this study we used site‐directed mutagenesis of the cDNA encoding PEDF to show that (a) truncation of the C‐terminal tail (Pro415–Pro418) of PEDF, (b) deletion of the Pro373–Ala380 segment that resides within the reactive center loop of the protein, and (c) alanine substitution of amino‐acid residues Asn391–Thr403 located within its hydrophobic core inhibit PEDF secretion, but not its transcription, by cells transfected with the various PEDF cDNAs. On the basis of the crystal structure of PEDF, these mutations are presumed to alter the protein conformation, suggesting that conservation of the 3D structure of PEDF is essential for its secretion. In addition, we show that replacement of Gly376 and Leu377 with alanine prevents PEDF secretion. As these two residues are located within the highly exposed segment of the reactive center loop, we propose a novel function for this loop in PEDF. Our results imply that the reactive center loop, specifically Gly376 and Leu377, is involved in the interaction of PEDF with components of the quality control system in the endoplasmic reticulum, thus ensuring its efficient secretion.
Mosquito (Aedes aegypti ) aquaporin, present in tracheolar cells, transports water, not glycerol, and forms orthogonal arrays in Xenopus oocyte membranes
Authors: Duchesne Laurence , Hubert Jean‐François , Verbavatz Jean‐Marc , Thomas Daniel , Pietrantonio Patricia V.
Published: March 2003

Previous results showed that mRNA encoding a putative aquaporin (AQP) (GenBank accession number AF218314) is present in the tracheolar cells associated with female Aedes aegypti Malpighian tubules. In this study, immunohistochemistry detected the protein, AeaAQP, also in tracheolar cells, suggesting its involvement in water movement in the respiratory system. When expressed in Xenopus oocytes, AeaAQP increased the osmotic water permeability from 15 × 10−6 to 150 × 10−6 m·s−1, which was inhibited by mercury ions. No permeability to glycerol or other solute was observed. AeaAQP expressed in oocytes was solubilized as a homotetramer in nondenaturing detergent as deduced from velocity centrifugation on density gradients. Phylogenetic analysis of MIP (major intrinsic protein) family sequences shows that AeaAQP clusters with other native orthogonal array forming proteins. Specific orthogonal arrays were detected by freeze‐fracture analysis of AeaAQP oocyte membranes. We conclude that, in tracheolar cells of A. aegypti, AeaAQP is probably a highly water‐permeable homotetrameric MIP which natively can form 2D crystals.
Assessment of telomere length and factors that contribute to its stability
Authors: Saldanha Sabita N. , Andrews Lucy G. , Tollefsbol Trygve O.
Published: March 2003

Short strands of tandem hexameric repeats known as telomeres cap the ends of linear chromosomes. These repeats protect chromosomes from degradation and prevent chromosomal end‐joining, a phenomenon that could occur due to the end‐replication problem. Telomeres are maintained by the activity of the enzyme telomerase. The total number of telomeric repeats at the terminal end of a chromosome determines the telomere length, which in addition to its importance in chromosomal stabilization is a useful indicator of telomerase activity in normal and malignant tissues. Telomere length stability is one of the important factors that contribute to the proliferative capacity of many cancer cell types; therefore, the detection and estimation of telomere length is extremely important. Until relatively recently, telomere lengths were analyzed primarily using the standard Southern blot technique. However, the complexities of this technique have led to the search for more simple and rapid detection methods. Improvements such as the use of fluorescent probes and the ability to sort cells have greatly enhanced the ease and sensitivity of telomere length measurements. Recent advances, and the limitations of these techniques are evaluated.

 Drugs that assist in telomere shortening may contribute to tumor regression. Therefore, factors that contribute to telomere stability may influence the efficiency of the drugs that have potential in cancer therapy. These factors in relation to telomere length are also examined in this analysis.
Novel aspects of calmodulin target recognition and activation
Authors: Vetter Stefan W. , Leclerc Estelle
Published: March 2003

Several crystal and NMR structures of calmodulin (CaM) in complex with fragments derived from CaM‐regulated proteins have been reported recently and reveal novel ways for CaM to interact with its targets. This review will discuss and compare features of the interaction between CaM and its target domains derived from the plasma membrane Ca2+‐pump, the Ca2+‐activated K+‐channel, the Ca2+/CaM‐dependent kinase kinase and the anthrax exotoxin. Unexpected aspects of CaM/target interaction observed in these complexes include: (a) binding of the Ca2+‐pump domain to only the C‐terminal part of CaM (b) dimer formation with fragments of the K+‐channel (c) insertion of CaM between two domains of the anthrax exotoxin (d) binding of Ca2+ ions to only one EF‐hand pair and (e) binding of CaM in an extended conformation to some of its targets. The mode of interaction between CaM and these targets differs from binding conformations previously observed between CaM and peptides derived from myosin light chain kinase (MLCK) and CaM‐dependent kinase IIα (CaMKIIα). In the latter complexes, CaM engulfs the CaM‐binding domain peptide with its two Ca2+‐binding lobes and forms a compact, ellipsoid‐like complex. In the early 1990s, a model for the activation of CaM‐regulated proteins was developed based on this observation and postulated activation through the displacement of an autoinhibitory or regulatory domain from the target protein upon binding of CaM. The novel structures of CaM‐target complexes discussed here demonstrate that this mechanism of activation may be less general than previously believed and seems to be not valid for the anthrax exotoxin, the CaM‐regulated K+‐channel and possibly also not for the Ca2+‐pump.