bluuebunny/crossref_metadata_2025 huggingface dataset:
A probability model for the meiosis I non‐disjunction fraction in numerical chromosomal anomalies
Authors: Franco Glaura C. , Lucio Paulo S. , Parra Flávia C. , Pena Sérgio D. J.
Published: June 2003

Numerical chromosome abnormalities (aneuploidies) are among the most common known causes of mental retardation and the leading cause of pregnancy loss in humans. They primarily arise by the process of meiotic non‐disjunction. We still know very little about the contribution of genetic and environmental causes for non‐disjunction in humans. In order to increase our understanding of the epi‐ demiology of human trisomies, it is necessary to establish the proportion of cases occurring in the first or second division of meiosis. Trisomic patients will display, in study of microsatellite typed by the polymerase chain reaction (PCR), three fragment peaks of equal intensity, two fragments at an average 2: 1 dosage or one individual fragment. In this work we describe a statistical approach for estimation of the fraction of meiosis I non‐disjunctions (F) in the absence of the parental information. First we determine a probability model for the number of peaks in a polymorphic microsatellite locus, which is a function of F. Based on this model, we obtain a maximum likelihood estimator for F, using the observed proportion of one, two and three allele patterns in trisomic individuals. Relying on the properties of maximum likelihood theory, we also calculate the asymptotic variance and confidence intervals for F. Owing to the fact that the samples of trisomic patients are limited in number, the use of asymptotic theory may be compromised. Thus, we employ the bootstrap technique to build confidence intervals for F and compare the results with those obtained from the normal theory. This estimator that dispenses the need to study parents opens the possibility of using archival material for comparative epidemiological studies of Down's syndrome and other aneuploidies. In this paper we propose a probability model to estimate the fraction of meiosis I non‐disjunction, F, by only using the proportion of allele patterns of trisomy individuals, while traditional methods require typing pericentromeric markers from those affected and their parents. Copyright © 2003 John Wiley & Sons, Ltd.
Power calculations for large multi‐arm placebo‐controlled studies of dichotomous outcomes
Authors: Hasselblad Vic , Allen Andrew S.
Published: June 2003

Several statistical methods can be used to test for differences in placebo‐controlled dichotomous multi‐arm studies. These tests include linear and quadratic trends, Dunnett's test, Hochberg's test, and the overall chi‐square test. We computed the power of these tests under a wide variety of alternatives. For two arms versus a control, the power was computed exactly. For more than two arms, the power was calculated from 100000 simulations per alternative. Against monotonic alternatives, the linear trend test tended to be superior. Against convex alternatives, the quadratic trend test tended to be superior. Against general alternatives, Dunnett's test, Hochberg's test, and the overall chi‐square test were superior and had very similar power curves. The Hochberg test was generally less powerful than Dunnett's test but the difference was offset by its increased power to reject multiple hypotheses. These results remained nearly constant over a wide range of sample sizes, average event rates and average effect sizes. Copyright © 2003 John Wiley & Sons, Ltd.
Three‐tier replication for FT‐CORBA infrastructures
Authors: Baldoni Roberto , Marchetti Carlo
Published: June 2003

Enforcing strong replica consistency among a set of replicas of a service deployed across anasynchronousdistributed system in the presence of crash failures is a real practical challenge. If each replica runs the consistency protocol bundled with the actual service implementation, this target cannot be achieved, as replicas need to be located over apartially synchronous distributed systemto solve the distributed agreement problems underlying strong replica consistency.

A three‐tier architecture for software replication enables the separation of the replication logic, i.e. protocols and mechanisms necessary for managing software replication, from both clients and server replicas. The replication logic is embedded in a middle‐tier that confines the need of partial synchrony and thus frees replica deployment.

In this paper we first introduce the basic concepts underlying three‐tier replication. Then we present the interoperable replication logic (IRL) architecture, a fault‐tolerant CORBA compliant infrastructure. IRL exploits a three‐tier approach to replicate stateful deterministic CORBA objects and allows object replicas to run on object request brokers from different vendors. A description of an IRL prototype developed in our department is proposed along with an extensive performance analysis. Copyright © 2003 John Wiley & Sons, Ltd.
Grid Integration of Wind Farms
Authors: Tande John Olav Giæver
Published: June 2003

This article gives an overview of grid integration of wind farms with respect to impact on voltage quality and power system stability. The recommended procedure for assessing the impact of wind turbines on voltage quality in distribution grids is presented. The procedure uses the power quality characteristic data of wind turbines to determine the impact on slow voltage variations, flicker, voltage dips and harmonics. The detailed assessment allows for substantially more wind power in distribution grids compared with previously used rule‐of‐thumb guidelines. Power system stability is a concern in conjunction with large wind farms or very weak grids. Assessment requires the use of power system simulation tools, and wind farm models for inclusion in such tools are presently being developed. A fixed‐speed wind turbine model is described. The model may be considered a good starting point for development of more advanced models, hereunder the concept of variable‐speed wind turbines with a doubly fed induction generator is briefly explained. The use of dynamic wind farm models as part of power system simulation tools allows for detailed studies and development of innovative grid integration techniques. It is demonstrated that the use of reactive compensation may relax the short‐term voltage stability limit and allow integration of significantly more wind power, and that application of automatic generation control technology may be an efficient means to circumvent thermal transmission capacity constraints. The continuous development of analysis tools and technology for cost‐effective and secure grid integration is an important aid to ensure the increasing use of wind energy. A key factor for success, however, is the communication of results and gained experience, and in this regard it is hoped that this article may contribute. Copyright © 2003 John Wiley & Sons, Ltd.
Developing wind energy to meet the Kyoto targets in the European union
Authors: Zervos Arthouros
Published: June 2003

Wind energy is the fastest‐growing electricity‐generating technology. The wind energy targets set during the last decade have all been surpassed. New targets are proposed for 2010 and 2020, since the existing European Commission and European Wind Energy Association targets do not reflect the trends in the market. The new targets and projections are used to define the role of wind energy in the electricity generation of the European Union during the next two decades. Estimation is made of the emission reductions which could be achieved with the use of wind energy by each Member State, and the contribution that wind energy could have in the process of the fulfilment of their Kyoto commitments is investigated. Finally an estimation of the costs involved is performed, with special reference to the avoided external costs. Copyright © 2003 John Wiley & Sons, Ltd.
Present Status of Aeroelasticity of Wind Turbines
Authors: Rasmussen Flemming , Hansen Morten Hartvig , Thomsen Kenneth , Larsen Torben Juul , Bertagnolio Franck , Johansen Jeppe , Madsen Helge Aagaard , Bak Christian , Hansen Anders Melchior
Published: June 2003

Aeroelasticity is a key issue in the continuing development of wind turbines towards large, flexible, highly optimized machines. The steadily increasing technology level is reflected not necessarily by the complexity of the turbines, but by the level of knowledge and advanced principles that are taken into account during the design process and incorporated in the final optimized design. This article examines some of the main uncertainties within aeroelastic modelling of wind turbines that have been hard to resolve over the years of research, and also presents perspectives for the new aeroelastic challenges, both from the viewpoint of the researchers at Risø National Laboratory. Copyright © 2003 John Wiley & Sons, Ltd.
Na+‐dependent fructose transport via rNaGLT1 in rat kidney
Authors: Horiba Naoshi , Masuda Satohiro , Ohnishi Chiharu , Takeuchi Daisuke , Okuda Masahiro , Inui Ken-ichi
Published: June 2003

We found a system of Na+‐dependent uptake of fructose by rat renal brush‐border membrane vesicles. It consisted of two saturable components, and was thought to involve at least two transporters. rNaGLT1, a novel glucose transporter in rat kidney, showed fructose uptake as well as α‐methyl‐D‐glucopyranoside uptake by transfected HEK293 cells. The features of the lower affinity type of fructose transporter in the brush‐border membranes, such as affinity and substrate recognition, were very comparable with those of rNaGLT1‐transfected HEK293 cells. These results indicated that rNaGLT1 is a primary fructose transporter in rat renal brush‐border membranes.
The diverse spectrum of sliding clamp interacting proteins
Authors: Vivona Jonathan B. , Kelman Zvi
Published: June 2003

DNA polymerase sliding clamps are a family of ring‐shaped proteins that play essential roles in DNA metabolism. The proteins from the three domains of life, Bacteria, Archaea and Eukarya, as well as those from bacteriophages and viruses, were shown to interact with a large number of cellular factors and to influence their activity. In the last several years a large number of such proteins have been identified and studied. Here the various proteins that have been shown to interact with the sliding clamps of Bacteria, Archaea and Eukarya are summarized.
The Status of Lecanora zosterae in the British Isles
Authors: Laundon Jack Rodney
Published: June 2003

Lecanora zosterae (Ach.) Nyl. is a distinct species belonging to the Lecanora dispersa group. In the British Isles it is confined to the coast where it grows mostly on the exposed turf above sea cliffs. Some 76% of the population occurs on decayed thrift Armeria maritima. The related Lecanora umbrina (Ach.) A. Massal. is lectotypified; it is the correct name for Lecanora hagenii auct.
Studies of new polysaccharides from lasallia pustulata (L.) Hoffm
Authors: Pereyra M. T. , Prieto A. , Bernabé M. , Leal J. A.
Published: June 2003

Polysaccharides from thalli of Lasallia pustulata were extracted and characterized. The major fractions were characterized as pustulan, a β-glucan-chitin complex and a β-(1→3)-glucan. Other polysaccharides, which are minor components (less than 5%) solubilized with water from the alkali extracts of thalli, were shown to be galactomannans with a backbone of α-(l→6)- mannose partially substituted at positions 2 by galactofuranose and/or mannopyranose side chains of variable length. The relationship between these polysaccharides and those obtained by the same method from free-living fungi is discussed. The proportion or composition of the different polysaccharides studied was not affected by seasonal environmental conditions over a 2-year period.
The Lichen genus Ramalina Ach. (Ramalinaceae) on the Outlying Islands of the New Zealand geographic area
Authors: Bannister J. M. , Blanchon D. J.
Published: June 2003

The diversity of species of Ramalina occurring on the outlying islands of the New Zealand geographic area is linked to their methods of dispersal and the origin and principal climatic features of the islands themselves. It appears that species of Ramalina have reached these islands by transoceanic, wind-borne dispersal of ascospores and soredia, not necessarily in the direction of the prevailing winds. Species have become established on the islands only if both climatic and habitat requirements have been met.
Spatial organization of three‐dimensional cocultures of adriamycin‐sensitive and ‐resistant human breast cancer MCF‐7 cells
Authors: Starzec A. , Briane D. , Kraemer M. , Kouyoumdjian J.‐C. , Moretti J.‐L. , Beaupain R. , Oudar O.
Published: June 2003

Genetic and cellular heterogeneity is one of mechanisms involved in increasing tumour aggressiveness during neoplastic progression. Development of drug‐resistant tumour cell subpopulations is a major problem in clinical oncology. Multi‐drug resistant tumour cells survive when exposed to cytotoxic agents. Here, we studied in a three‐dimensional (3D) coculture system, called “ex vivo nodules”, how drug‐resistant and sensitive tumour cells settle down in a 3D space. For this, we cocultured adriamycin‐sensitive (MCF‐7S) and ‐resistant (MCF‐7R) human breast cancer cells in long term nodules. We showed that both types of cells are able to grow separately or in coculture until five weeks in spheroidal forms. MCF‐7R cells did not loose their multi‐drug resistance when cultured in nodules as measured by RT‐PCR. Curiously, the exterior aspects of mixed (MCF‐7S/ MCF‐7R) nodules and MCF‐7R nodules were similar whereas MCF‐7S nodules were completely different. Nevertheless, morphologically these three nodule types were distinct, in particular in their density. Immunostaining showed that in mixed nodules, MCF‐7R cells were arranged at the periphery, whereas the MCF‐7S cells are in the central part of the nodules.

Even if the mechanism of this arrangement remained unclear, this work shows that three‐dimensional cell culture is well adapted to the study of the relationships between adhesion mechanisms and drug‐resistance.
Ascidians as a vertebrate‐like model organism for physiological studies of Rho GTPase signaling
Authors: Philips Alexandre , Blein Marion , Robert Agnès , Chambon Jean‐Philippe , Baghdiguian Stephen , Weill Mylène , Fort Philippe
Published: June 2003

GTPases of the Rho family are evolutionarily conserved proteins that control cell shape dynamics during physiological processes as diverse as cell migration and polarity, axon outgrowth and guidance, apoptosis and phagocytosis. In mammals, 18 Rho proteins are distributed in 7 subfamilies. Rho, Rac and Cdc42 are the best‐characterized ones, benefiting from the use of worm and drosophila, which only express these 3 subfamilies. An additional model would therefore help understand the physiological role of other mammalian subfamilies. We identified in genome databases the complete Rho family of two ascidians, Ciona intestinalis and Ciona savignyi, and showed that these families contain single ancestors of most mammalian Rho subfamilies. In Ciona intestinalis, all Rho genes are expressed and display specific developmental variations of mRNA expression during tadpole formation. Although C. intestinalis expresses five additional Rac compared to the closely related Ciona savignyi, only two appeared fully active in functional assays. Last, we identified in Ciona intestinalis database more than 50 Rho regulators (RhoGEFs and RhoGAPs) and 20 effector targets, whose analysis further supports the notion that Rho signaling components are of comparable complexity in mammals and ascidians. Since the tadpole of ascidians combines vertebrate‐like developmental features with reduced cell number, particularly adapted to evolutionary and developmental biology studies, our data advocate this model for physiological studies of Rho signaling pathways.
Misexpression of mouse porcupine isoforms modulates the differentiation of P19 embryonic carcinoma cells
Authors: Tanaka Kimiko , Kitagawa Yasuo , Kadowaki Tatsuhiko
Published: June 2003

Drosophila porcupine (porc) encodes an ER membrane protein that is required for the processing of the Drosophila Wnt family. Homologs of porc have been identified in various multicellular organisms and have been implicated in the biosynthesis of Wnt proteins. In contrast to Drosophila, vertebrates generate four different porc mRNAs (A—D) by alternative splicing. Murine porcD (MporcD) mRNA levels transiently increase during the neuroectodermal differentiation of P19 cells, but diminish during mesodermal differentiation. P19 cells constitutively expressing mouse porcA (MporcA), but not MporcD, undergo apoptosis by the induction of neuroectodermal differentiation. Meanwhile, P19 cells constitutively expressing MporcD, but not MporcA, do not adopt mesodermal cell morphology and fail to express myf‐5 when induced to mesodermal differentiation. These results therefore demonstrate that the alternative splicing of Mporc is regulated in a cell‐type specific manner, and the resulting Mporc isoforms have different functions in the neuroectodermal and mesodermal differentiation of P19 cells.
Haemorrhage in seven cats with suspected anticoagulant rodenticide intoxication
Authors: Kohn B , Weingart C , Giger U
Published: June 2003

Clinical features were evaluated in seven adult cats (six males, one female) with haemorrhage and presumptive anticoagulant rodenticide intoxication. Haemorrhage appeared as thoracic haemorrhage, otic bleeding, haematoma, melena, haematochezia, and petechiation. The most common other presenting signs were lethargy, anorexia, and tachypnoea or dyspnoea. Six cats were anaemic, four cats were mildly thrombocytopenic (58 000–161 000/μl), and three had slightly decreased plasma protein or albumin values. The prothrombin time (30.3–>100 s, reference range: 16.5–27.5 s) and activated partial thromboplastin time values (32.6–>100 s; reference range: 14–25 s) were markedly prolonged in all cats. All cats received vitamin K1 subcutaneously or orally (3.7–5 mg/kg body weight initially) and depending on severity of signs five cats were transfused with fresh whole blood. Plasma coagulation times improved in all cats and returned to normal in 1–5 days. Rodenticide poisons represent an important but relatively rare cause of haemorrhage in cats and can be effectively treated.
Clinical features and contemporary management of patients with low and preserved ejection fraction heart failure: baseline characteristics of patients in the Candesartan in Heart failure—Assessment of Reduction in Mortality and morbidity (CHARM) programme
Authors: McMurray J. , Östergren J. , Pfeffer M. , Swedberg K. , Granger C. , Yusuf S. , Held P. , Michelson E. , Olofsson B. ,   On behalf of the CHARM committees and investigators
Published: June 2003

Aims:

To describe the clinical characteristics and contemporary treatment of a broad spectrum of patients with chronic heart failure (CHF) randomised in the Candesartan in Heart failure—Assessment of Reduction in Mortality and morbidity (CHARM) programme, consisting of three component studies comparing placebo to candesartan.

Methods and results:

CHARM Alternative, CHARM Added and CHARM Preserved enrolled 2028 low left ventricular ejection fraction (LVEF) ACE inhibitor intolerant patients, 2548 low LVEF ACE inhibitor treated patients and 3025 preserved LVEF patients, respectively. Patients in CHARM Preserved were more often female. The proportion of women in CHARM Preserved was 40% compared to 32% in CHARM Alternative and 21% in CHARM Added. Patients in CHARM Preserved were also more often hypertensive than in the other two trials (64% vs. 50% and 48%, respectively). Symptoms and signs (with the exception of a third heart sound) were similar in all three patient groups. Beta‐blockers were used in over half of patients in all three groups. Digoxin and spironolactone were used less frequently and calcium antagonists more frequently in CHARM Preserved. Spironolactone was used most frequently in CHARM Alternative, i.e. in ACE inhibitor intolerant patients.

Conclusions:

The CHARM Programme provides the largest and most detailed comparison to date of patients low‐ and preserved‐LVEF CHF. It also describes the causes of ACE‐inhibitor intolerance in a large cohort of patients and the other treatment which these patients receive.
Update of clinical trials from the American College of Cardiology 2003. EPHESUS, SPORTIF‐III, ASCOT, COMPANION, UK‐PACE and T‐wave alternans
Authors: Cleland John G.F. , Coletta Alison P. , Nikitin Nikolay , Louis Amala , Clark Andrew
Published: June 2003

The American College of Cardiology provided much useful new information to inform those who care for patients with heart failure about what they should and should not adopt into current clinical practice. The EPHESUS trial suggests a much wider role for aldosterone antagonists for the management of heart failure and left ventricular systolic dysfunction. SPORTIF‐III indicates we may have a safer, simpler warfarin substitute soon. ASCOT reinforces the potential futility of statin therapy unless it is well targeted. The results of the COMPANION study investigating cardiac resynchronisation devices and implantable defibrillators were encouraging but inconclusive and/or hard to interpret. UK‐PACE again questions the use of dual chamber pacing. T‐wave alternans is an interesting experimental technique that may be useful in selecting which patients need an implantable defibrillator, although the technology needs testing in an appropriate patient population.
Introduction: International Symposium on Modern Trends in Malaria
Authors: Pasternak Charles A.
Published: June 2003

Malaria continues to kill some two million people a year, half of whom are young children. We have no vaccine, the parasite has become resistant to the most effective drugs, and elimination of the mosquito vector through spraying with insecticide is being questioned. Yet research on malaria is — at last — being funded reasonably well. What has been achieved?

Scientists gathered at the All India Institute of Medical Sciences (AIIMS) in New Delhi from 13-15 February 2003 to discuss these issues. Because many antima-larial strategies work at the cell surface (see the abstract on Membranes as future therapeutic targets) it seems appropriate to give readers of Bioscience Reports: Molecular and Cellular Biology of the Cell Surface an opportunity to catch a glimpse of the current situation through the (unedited) abstracts of the invited speakers.
Attitudes of Health Care Trainees About Genetics and Disability: Issues of Access, Health Care Communication, and Decision Making
Authors: Ormond Kelly E. , Gill Carol J. , Semik Patrick , Kirschner Kristi L.
Published: June 2003

Prior studies suggest that knowledge and attitudes of health care professionals influence patient communication and medical decision making. To study this dynamic in the context of genetic disability, we developed a survey on health professionals' attitudes regarding disability and genetic screening and pilot‐tested it on a sample of medical students, residents, and genetic counseling students (N = 85). Despite minimal experience with disability or genetics, most respondents reported feeling comfortable dealing with genetics (59%) and disability (75%). The majority felt that disability caused significant suffering for both the person (51%) and family (64%), and that research should be directed toward preventing genetic disability (62%). Similar to prior literature, perceived “Quality of Life” was most often based on degrees of physical and cognitive functioning, pain, and social support. However, differences were found between genetic counseling trainees and other medical trainees in their relative emphasis of social versus medical issues in questions of disability and genetic testing, and these response patterns were associated with differences in the groups' priorities for offering information about social resources. Respondents agreed that access to genetic testing and information is personal and that testing should be available upon request for oneself (68%) and to a lesser degree for one's fetus (55%) or child (41%). However, the same individuals frequently stated that society should regulate access to such technologies. Although most felt that the patient and professional should jointly make such decisions on a case‐by‐case basis, it was also seen as appropriate for the health care professional to occasionally decline genetic testing. It seems apparent that training and experience influence knowledge and attitudes. Therefore, it is critical to document knowledge and attitudes of various health care providers and trainees, including differences between various specialties, to improve educational interventions geared to this area.
Community Science: Bridging the Gap between Science and Practice with Community‐Centered Models
Authors: Wandersman Abraham
Published: June 2003

A major goal of community science is to improve the quality of life in our communities by improving the quality of the practice of treatment, prevention, health promotion, and education. Community science is an interdisciplinary field, which develops and researches community‐centered models that enable communities to use evidence‐based interventions more effectively and efficiently. In this article, the gap between science and practice and the need to bridge the gap with new models serve as an entry point and guide to the development of a community science. Therefore, the article describes (1) the “prevention science” model of bringing science to practice, (2) why this model is necessary but not sufficient for influencing the quality of interventions in our everyday world, (3) the gap between science and practice and the need to integrate “prevention science” models with community‐centered models in order to bridge the gap, and (4) features of community science.
Developing Standard Recommendations (Guidelines) for Genetic Counseling Practice: A Process of the National Society of Genetic Counselors
Authors: Bennett Robin L. , Pettersen Barbara J. , Niendorf Kristin B. , Anderson Rebecca Rae
Published: June 2003

The National Society of Genetic Counselors (NSGC) supports the development of practice recommendations (guidelines) in the field of genetic counseling. This paper reviews the basic components of NSGC genetic counseling practice recommendations as well as the process for formal adoption of such documents, as approved by the Board of Directors of the NSGC.
Design and performance of a tunable diode laser absorption spectrometer for airborne formaldehyde measurements
Authors: Wert B. P. , Fried A. , Rauenbuehler S. , Walega J. , Henry B.
Published: June 2003

A tunable diode laser absorption spectrometer (TDLAS) was modified for high‐precision and high‐time‐resolution formaldehyde (CH2O) measurements. This enhanced system was deployed in both the clean and polluted troposphere, as part of aircraft missions (TOPSE 2000, TexAQS 2000, and TRACE‐P 2001) and ground‐based missions (SOS 1999). Measurements of very constant ambient CH2O concentrations were used to determine instrument precisions, which were stable under normal operating conditions, with the exception of brief aircraft cabin pressure changes. Precisions of 15–50 pptv (1σ) were typically achieved for 1 min of averaging, corresponding to absorptions of 0.5–1.7 × 10−6, 3–5 times better than the previous version of the instrument (1998). Responsible modifications included improved temperature and pressure control of instrument components and the use of more stable optical mounts. During the TexAQS 2000 aircraft mission (polluted continental troposphere), measurements of 1 s time resolution were reported. Instrument accuracy was validated by calibration cross checks, interference tests, sample transmission tests, and field comparisons with a DOAS system.
Pi2 pulsations with second harmonic: CRRES observations in the plasmasphere
Authors: Takahashi Kazue , Anderson Roger R. , Hughes W. Jeffrey
Published: June 2003

Pi2 pulsations observed on the ground at low latitudes often exhibit multiple spectral peaks, and this property has been attributed to the harmonics of plasmaspheric cavity mode resonance. We show that multifrequency Pi2 pulsations simultaneously observed from the Combined Release and Radiation Effects Satellite (CRRES) and at Kakioka (L = 1.26) can be explained by the cavity mode. The pulsations occurred in a 40‐min time span on 20 January 1991. CRRES was within the plasmasphere at L = 6.1, on the dipole equator, and very near midnight. Kakioka was also very close to midnight. The pulsations consisted of two spectral components, ∼7 mHz (f1, fundamental) and ∼12 mHz (f2, second harmonic). At CRRES, the f1 oscillation was observed in the azimuthal component, Eφ, of the electric field, while the f2 oscillation was observed in the compressional component, Bz, of the magnetic field. At Kakioka, by contrast, both the fundamental and second harmonic oscillations were detected in the horizontal component, H. The spectral separation between Eφ and Bz implies that the spacecraft was located near the node/antinode of cavity mode resonance excited at multiple harmonics. Further support for this interpretation is provided from analyses of the phase of Eφ and Bz.
Movement of Plasma‐Membrane‐Associated Clathrin Spots Along the Microtubule Cytoskeleton
Authors: Rappoport Joshua Z. , Taha Bushra W. , Simon Sanford M.
Published: June 2003

The current understanding of the role of plasma‐ membrane‐associated clathrin suggests that clathrin‐coated pits form at the sites of activated receptors and then, following internalization, the clathrin coat is rapidly shed. Utilizing total internal reflection fluorescence microscopy (TIR‐FM), we have documented linear lateral motion of cell‐surface‐associated dsRed‐clathrin spots parallel to the plasma membrane. Clathrin spot motility was observed in multiple cell lines (MDCK, CHO, Cos‐7 and HeLa). In MDCK cells dsRed‐clathrin spots moved along linear pathways up to 4 μm in length with rates of approximately 0.8 μm/s. Spots did not generally undergo internalization during movement. The motion of these puncta was coincident with the microtubule cytoskeleton, and depolymerization of microtubules reduced spot motility over 10‐fold. Over‐expression of the microtubule‐associated protein tau‐EGFP decreased spot run length by 40% without affecting the rate of movement. Thus dsRed‐clathrin puncta move along the microtubule cytoskeleton parallel to the cell surface.
Elucidation of Basal Body and Centriole Functions in Chlamydomonas reinhardtii
Authors: Dutcher Susan K.
Published: June 2003

In eukaryotic cells, basal bodies and their structural equivalents, centrioles, play essential roles. They are needed for the assembly of flagella or cilia as well as for cell division. Chlamydomonas reinhardtii provides an excellent model organism for the study of the basal body and centrioles. Genes for two new members of the tubulin superfamily are needed for basal body/centriole duplication. In addition, other genes that play roles in the duplication and segregation of basal bodies are discussed.
Protein Transport and Signal Transduction During Fertilization in Chlamydomonas
Authors: Pan Junmin , Misamore Michael J. , Wang Qian , Snell William J.
Published: June 2003

Fertilization in Chlamydomonas begins with flagellar adhesion between mating type plus and mating type minus gametes and is consummated within minutes by zygote formation. Once fusion occurs, the newly merged gametes cease existence as distinct entities, and the diploid zygote immediately initiates transcription of zygote‐specific genes. Accomplishing fertilization within such a short time requires the rapid and signaled movement of pre‐existing membrane and cytoplasmic proteins between and within several cellular compartments. Generation within the adhering flagella of the initial signals for protein movement, as well as movement itself of at least one cytoplasmic protein from the cell body to the flagella, depend on the microtubule motor, kinesin‐II and presumably on intraflagellar transport (IFT). Adhesion and fusion of the two gametes depend on a second translocation event, the movement of an adhesion/fusion protein onto the surface of a rapidly elongating, microvillous‐like fusion organelle. Finally, the merging of the two separate gametes, each containing sex‐specific proteins, into a single cell allows the formerly separate proteins to form new interactions that regulate zygote development. Two proteins – a nuclease and a homeodomain protein – which were present only in the plus gamete, are ‘delivered’ to the cytoplasm of the zygote during gamete fusion. The nuclease is selectively imported into the minus chloroplast, where it degrades the chloroplast DNA, thereby ensuring uniparental inheritance of plus chloroplast traits. The homeodomain protein binds with an as yet unidentified protein delivered by the minus gamete, and the new complex activates transcription of zygote‐specific genes.
PtdIns(3,5)P2 is Required for Delivery of Endocytic Cargo into the Multivesicular Body
Authors: Shaw Jonathan D. , Hama Hiroko , Sohrabi Farrokh , DeWald Daryll B. , Wendland Beverly
Published: June 2003

The endocytic pathway transports cargo from the plasma membrane to early endosomes, where certain cargoes are sorted to the late endosome/multivesicular body. Biosynthetic cargo destined for the lysosome is also trafficked through the multivesicular body. Once delivered to the multivesicular body, cargo destined for the interior of the lysosome is selectively sorted into vesicles that bud into the lumen of the multivesicular body. These vesicles are released into the lumen of the lysosome upon the fusion of the multivesicular body and lysosomal limiting membranes. The yeast protein Fab1, which catalyzes the production of phosphatidylinositol (3,5) bisphosphate [PtdIns(3,5)P2], is necessary for proper sorting of biosynthetic cargo in the multivesicular body. Utilizing an endocytosis screen, we isolated a novel allele of FAB1 that contains a point mutation in the lipid kinase domain. Characterization of this allele revealed reduced PtdIns(3,5)P2 production, altered vacuole morphology, and biosynthetic protein sorting defects. We also found that endocytosis of the plasma membrane protein Ste3 is partially blocked downstream of the internalization step, and that delivery of the dye FM4‐64 to the vacuole is delayed in fab1 mutants. Additionally, Ste3 is not efficiently sorted into multivesicular body vesicles in fab1 mutants and instead localizes to the vacuolar limiting membrane. These data show that PtdIns(3,5)P2 is necessary for proper trafficking and sorting of endocytic cargo through the late endosome/multivesicular body.
Construction, expression and characterization of a chimaeric mammalian-plant aspartic proteinase
Authors: PAYIE Kenneth G. , TANAKA Takuji , GAL Susannah , YADA Rickey Y.
Published: June 2003

Aspartic proteinases are a well-characterized class of proteinases. In plants, all nascent aspartic proteinases possess a 100-amino-acid, plant-specific sequence (PSS) within their C-terminal lobe, presumed to possess a targeting role in vivo. In this study, the PSS domain from the Arabidopsis thaliana aspartic proteinase was inserted into porcine pepsinogen at the identical location found in nascent plant aspartic proteinases, to create a chimaeric mammalian–plant enzyme. Based on enzymic activity, this chimaeric enzyme demonstrated increases in pH stability above 6 and temperature stability above 60 °C compared with commercial pepsin. Differential scanning calorimetry of the chimaeric enzyme illustrated an approx. 2 °C increase in denaturation temperature (Tm), with increases in co-operativity and similar enthalpy values. Kinetic analysis indicated an increase in Km and decreased kcat compared with pepsin, but with a catalytic efficiency similar to the monomeric plant aspartic proteinase from wheat. Using oxidized insulin B-chain, the chimaeric enzyme demonstrated more restricted substrate specificity in comparison with commercial pepsin. This study highlights the use of a chimaeric enzyme strategy in order to characterize unique protein domains within enzyme families, and, for the first time, a putative structure–function role for the PSS as it pertains to plant aspartic proteinases.
Haem and nitric oxide: synergism in the modulation of the endothelial haem oxygenase-1 pathway
Authors: FORESTI Roberta , HOQUE Martha , BAINS Sandip , GREEN Colin J. , MOTTERLINI Roberto
Published: June 2003

NO potently up-regulates vascular haem oxygenase-1 (HO-1), an inducible defensive protein that degrades haem to CO, iron and the antioxidant bilirubin. Since several pathological states are characterized by increased NO production and liberation of haem from haem-containing proteins, we examined how NO influences HO-1 induction mediated by haemin. Aortic endothelial cells treated with S-nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside (SNP) or diethylenetriamine-NONOate (DETA/NO) and haemin exhibited higher levels of haem oxygenase activity compared with cells exposed to NO donors or haemin alone. This was accompanied by a marked increase in bilirubin production and, notably, by a strong magnification of cellular haem uptake. A role for haem metabolites in modulating HO-1 expression by NO was assessed by exposing cells to SNAP, SNP or DETA/NO in medium derived from cells treated with haemin, which contained increased bilirubin levels. This treatment considerably potentiated HO-1 expression and haem oxygenase activity mediated by NO and the use of a haem oxygenase inhibitor abolished this effect. Both iron liberated during haem breakdown and the formation of nitroxyl anion from NO appeared to partially contribute to the amplifying phenomenon; in addition, medium from haemin-treated cells significantly augmented the release of NO by NO donors. Thus we have identified novel mechanisms related to the induction of HO-1 by NO indicating that the signalling actions of NO vary significantly in the presence of haem and haem metabolites, ultimately increasing the defensive abilities of the endothelium to counteract oxidative and nitrosative stress.
Sequence motifs of tissue inhibitor of metalloproteinases 2 (TIMP-2) determining progelatinase A (proMMP-2) binding and activation by membrane-type metalloproteinase 1 (MT1-MMP)
Authors: WORLEY Joanna R. , THOMPKINS Philip B. , LEE Meng H. , HUTTON Mike , SOLOWAY Paul , EDWARDS Dylan R. , MURPHY Gillian , KNÄUPER Vera
Published: June 2003

Fundamental cellular processes including angiogenesis and cell migration require a proteolytic cascade driven by interactions of membrane-type matrix metalloproteinase 1 (MT1-MMP) and progelatinase A (proMMP-2) that are dependent on the presence of tissue inhibitor of metalloproteinases 2 (TIMP-2). There are unique interactions between TIMP-2 and MT1-MMP, which we have previously defined, and here we identify TIMP-2 sequence motifs specific for proMMP-2 binding in the context of its activation by MT1-MMP. A TIMP-2 mutant encoding the C-terminal domain of TIMP-4 showed loss of proMMP-2 activation, indicating that the C-terminal domain of TIMP-2 is important in establishing the trimolecular complex between MT1-MMP, TIMP-2 and proMMP-2. This was confirmed by analysis of a TIMP-4 mutant encoding the C-terminal domain of TIMP-2, which formed a trimolecular complex and promoted proMMP-2 processing to the intermediate form. Mutants encoding TIMP-4 from Cys1 to Leu185 and partial tail sequence of TIMP-2 showed some gain of activating capability relative to TIMP-4. The identified residues were subsequently mutated in TIMP-2 (E192–D193 to I192–Q193) and this inhibitor showed a significantly reduced ability to facilitate proMMP-2 processing by MT1-MMP. Furthermore, the tail-deletion mutant Δ186-194TIMP-2 was completely incapable of promoting proMMP-2 activation by MT1-MMP. Thus the C-terminal tail residues of TIMP-2 are important determinants for stable trimolecular complex formation between TIMP-2, proMMP-2 and MT1-MMP and play an important role in MT1-MMP-mediated processing to the intermediate and final active forms of MMP-2 at the cell surface.
Structural studies on the reaction of isopenicillin N synthase with the substrate analogue delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-alpha-aminobutyrate
Authors: LONG Alexandra J. , CLIFTON Ian J. , ROACH Peter L. , BALDWIN Jack E. , SCHOFIELD Christopher J. , RUTLEDGE Peter J.
Published: June 2003

Isopenicillin N synthase (IPNS) is a non-haem iron(II) oxidase which catalyses the biosynthesis of isopenicillin N from the tripeptide δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV). Herein we report crystallographic studies to investigate the reaction of IPNS with the truncated substrate analogue δ-(l-α-aminoadipoyl)-l-cysteinyl-d-α-aminobutyrate (ACAb). It has been reported previously that this analogue gives rise to three β-lactam products when incubated with IPNS: two methyl penams and a cepham. Crystal structures of the IPNS–Fe(II)–ACAb and IPNS–Fe(II)–ACAb–NO complexes have now been solved and are reported herein. These structures and modelling studies based on them shed light on the diminished product selectivity shown by IPNS in its reaction with ACAb and further rationalize the presence of certain key residues at the IPNS active site.
Structural analysis of xylanase inhibitor protein I (XIP-I), a proteinaceous xylanase inhibitor from wheat (Triticum aestivum, var. Soisson)
Authors: PAYAN Françoise , FLATMAN Ruth , PORCIERO Sophie , WILLIAMSON Gary , JUGE Nathalie , ROUSSEL Alain
Published: June 2003

A novel class of proteinaceous inhibitors exhibiting specificity towards microbial xylanases has recently been discovered in cereals. The three-dimensional structure of xylanase inhibitor protein I (XIP-I) from wheat (Triticum aestivum, var. Soisson) was determined by X-ray crystallography at 1.8 Å (1 Å=0.1 nm) resolution. The inhibitor possesses a (β/α)8 barrel fold and has structural features typical of glycoside hydrolase family 18, namely two consensus regions, approximately corresponding to the third and fourth barrel strands, and two non-proline cis-peptide bonds, Ser36–Phe and Trp256–Asp (in XIP-I numbering). However, detailed structural analysis of XIP-I revealed several differences in the region homologous with the active site of chitinases. The catalytic glutamic acid residue of family 18 chitinases [Glu127 in hevamine, a chitinase/lysozyme from the rubber tree (Hevea brasiliensis)] is conserved in the structure of the inhibitor (Glu128), but its side chain is fully engaged in salt bridges with two neighbouring arginine residues. Gly81, located in subsite −1 of hevamine, where the reaction intermediate is formed, is replaced by Tyr80 in XIP-I. The tyrosine side chain fills the subsite area and makes a strong hydrogen bond with the side chain of Glu190 located at the opposite side of the cleft, preventing access of the substrate to the catalytic glutamic acid. The structural differences in the inhibitor cleft structure probably account for the lack of activity of XIP-I towards chitin.
Structural-functional characterization of the cathodic haemoglobin of the conger eel Conger conger: molecular modelling study of an additional phosphate-binding site
Authors: PELLEGRINI Mariagiuseppina , GIARDINA Bruno , VERDE Cinzia , CARRATORE Vito , OLIANAS Alessandra , SOLLAI Luigi , SANNA Maria T. , CASTAGNOLA Massimo , PRISCO Guido di
Published: June 2003

The Conger conger (conger eel) haemoglobin (Hb) system is made of three components, one of which, the so-called cathodic Hb, representing approx. 20% of the total pigment, has been purified and characterized from both a structural and functional point of view. Stripped Hb showed a reverse Bohr effect, high oxygen affinity and slightly low cooperativity in the absence of any effector. Addition of saturating GTP strongly influences the pH dependence of the oxygen affinity, since the reverse Bohr effect, observed under stripped conditions, is converted into a small normal Bohr effect. A further investigation of the GTP effect on oxygen affinity, carried out by fitting its titration curve, demonstrated the presence of two independent binding sites. Therefore, on the basis of the amino acid sequence of the α- and β-chains, which have been determined, a computer modelling study has been performed. The data suggest that C. conger cathodic Hb may bind organic phosphates at two distinct binding sites located along the central cavity of the tetramer by hydrogen bonds and/or electrostatic interactions with amino acid residues of both chains, which have been identified. Among these residues, the two Lys-α(G6) (where the letter refers to the haemoglobin helix and the number to the amino acid position in the helix) appear to have a key role in the GTP movement from the external binding region to the internal central cavity of the tetrameric molecule.
The structure and regulation of the human and mouse matrix metalloproteinase-21 gene and protein
Authors: MARCHENKO George N. , MARCHENKO Natalia D. , STRONGIN Alex Y.
Published: June 2003

Matrix metalloproteinases (MMPs) play key roles in tissue remodelling under normal development and, especially, in diseases ranging from malignancies to stroke. We cloned and thoroughly characterized the novel human and mouse MMP gene encoding MMP-21. MMP-21 is the last uncharacterized MMP coded by the human genome. Human and mouse MMP-21 is the orthologue of Xenopuslaevis X-MMP. The latent proenzyme of MMP-21 (569 amino acid residues) consists of the prodomain, the catalytic domain and the haemopexin-like domain, and is potentially capable of being activated in its secretory pathway to the extracellular milieu by furin-like proprotein convertases. Human MMP-21 is the probable target gene of the Wnt pathway. In addition, the expression of MMP-21 is controlled uniquely by Pax and Notch transcription factors known to be critical for organogenesis. MMP-21 is expressed transiently in mouse embryogenesis and increased in embryonic neuronal tissues. Our observations clearly indicate that there is an important specific function for MMP-21 in embryogenesis, especially in neuronal cells.
Alternative splicing variants of dual specificity tyrosine phosphorylated and regulated kinase 1B exhibit distinct patterns of expression and functional properties
Authors: LEDER Susanne , CZAJKOWSKA Hanna , MAENZ Barbara , de GRAAF Katrin , BARTHEL Andreas , JOOST Hans-Georg , BECKER Walter
Published: June 2003

The dual specificity tyrosine phosphorylated and regulated kinase (DYRK) family of protein kinases is a group of evolutionarily conserved protein kinases that have been characterized as regulators of growth and development in mammals, Drosophila and lower eukaryotes. In the present study, we have characterized three splicing variants of DYRK1B (DYRK1B-p65, DYRK1B-p69 and DYRK1B-p75) with different expression patterns and enzymic activities. DYRK1B-p65 and DYRK1B-p69 exhibited similar, but not identical, patterns of expression in mouse tissues, with the highest protein levels found in the spleen, lung, brain, bladder, stomach and testis. In contrast, DYRK1B-p75 was detected specifically in skeletal muscles, in the neuronal cell line GT1-7 and also in differentiated, adipocyte-like 3T3-L1 cells, but not in undifferentiated 3T3-L1 preadipocytes. A comparison of the mouse and human Dyrk1b genomic and cDNA sequences defined the alternative splicing events that produce the variants of DYRK1B. In DYRK1B-p75, transcription starts with exon 1B instead of exon 1A, generating a new translation start, which extends the open reading frame by 60 codons. This gene structure suggests that alternative promoters direct the expression of DYRK1B-p69 and DYRK1B-p75. Both splicing variants exhibited kinase activity in vitro and contained phosphotyrosine when expressed in COS-7 cells. Owing to differential recognition of the 3′-splice site in exon 9, DYRK1B-p65 differs from DYRK1B-p69 by the absence of 40 amino acids within the catalytic domain. DYRK1B-p65 lacked kinase activity in vitro and did not contain phosphotyrosine. DYRK1B-p69 and DYRK1B-p75 stimulated reporter gene activity driven by the forkhead in rhabdosarcoma (FKHR)-dependent glucose-6-phosphatase promoter more strongly when compared with DYRK1B-p65, indicating that the DYRK1B splicing variants exhibit functional differences.