bluuebunny/crossref_metadata_2025 huggingface dataset:
Accelerated oxygen consumption in eastern Mediterranean deep waters following the recent changes in thermohaline circulation
Authors: Klein Birgit , Roether Wolfgang , Kress Nurit , Manca Beniamino Bruno , Ribera d'Alcala Maurizio , Souvermezoglou Ekaterini , Theocharis Alexander , Civitarese Guiseppe , Luchetta Anna
Published: September 2003

Using an intercalibrated set of oxygen data for the eastern Mediterranean, 1987–1999, we study the evolution of oxygen concentrations that accompanied the recent changes in the thermohaline circulation of this sea (the so‐called Eastern Mediterranean Transient (EMT)). We find that, by way of massively transferring oxygen‐rich near‐surface waters into the deep layers, the EMT by 1995 had raised oxygen concentrations considerably relative to the pre‐EMT situation in 1987. Between 1995 and 1999, however, the oxygen concentrations decreased noticeably, we identify oxygen decreases of about 5 μmol/kg for the layers below 2200 m and between about 500 and 1000 m depth, for which layers previous work found vanishing or little replenishment during 1995–1999. Supporting evidence for absence of replenishment is obtained from the temporal evolution of tritium‐3He ages. The oxygen decreases convert into utilization rates of approximately 1.3 μmol/(kg yr). An even higher rate, 2.3 μmol/(kg yr), is obtained for the deep waters of the Cretan Sea below 1000 m. The utilization rate found for the deepest waters significantly exceeds the pre‐EMT value of 0.53 μmol/(kg yr). We propose that the massive invasion of near surface waters affected by the EMT made available large amounts of dissolved organic carbon with an unusually high fraction of labile material, which in turn enhanced oxygen consumption. Supporting evidence is obtained from data of dissolved organic carbon, and from mesozooplankton ecology data. The enhanced oxygen utilization represents a further example of EMT‐related disturbances in the biogeochemistry of the eastern Mediterranean.
Photochemical production and evolution of selected C2–C5 alkyl nitrates in tropospheric air influenced by Asian outflow
Authors: Simpson Isobel J. , Blake Nicola J. , Blake Donald R. , Atlas Elliot , Flocke Frank , Crawford James H. , Fuelberg Henry E. , Kiley Christopher M. , Meinardi Simone , Rowland F. Sherwood
Published: September 2003

The photochemical production and evolution of six C2–C5 alkyl nitrates (ethyl‐, 1‐propyl‐, 2‐propyl‐, 2‐butyl‐, 2‐pentyl‐, and 3‐pentyl nitrate) was investigated using selected data from 5500 whole air samples collected downwind of Asia during the airborne Transport and Chemical Evolution over the Pacific (TRACE‐P) field campaign (February–April 2001). Air mass age was important for selecting appropriate field data to compare with laboratory predictions of C5 alkyl nitrate production rates. In young, highly polluted air masses, the ratio between the production rates of 3‐pentyl nitrate and 2‐pentyl nitrate from n‐pentane was 0.60–0.65. These measured ratios show excellent agreement with results from a field study in Germany (0.63 ± 0.06), and they agree better with predicted ratios from older laboratory kinetic studies (0.63–0.66) than with newer laboratory results (0.73 ± 0.08). TRACE‐P samples that did not show influence from marine alkyl nitrate sources were used to investigate photochemical alkyl nitrate evolution. Relative to 2‐butyl nitrate/n‐butane, the measured ratios of ethyl nitrate/ethane and 2‐propyl nitrate/propane showed notable deviations from modeled values based on laboratory kinetic data, suggesting additional Asian sources of their alkyl peroxy radical precursors. By contrast, the measured ratios of 1‐propyl‐, 2‐pentyl‐, and 3‐pentyl nitrate to their respective parent hydrocarbons were fairly close to modeled values. The 1‐propyl nitrate findings contrast with field studies in North America, and suggest that air downwind of Asia was not significantly impacted by additional 1‐propyl nitrate precursors. The sensitivity of modeled photochemical processing times to hydroxyl radical concentration, altitude, city ventilation times, and dilution is discussed.
Lower troposphere refractivity bias in GPS occultation retrievals
Authors: Ao C. O. , Meehan T. K. , Hajj G. A. , Mannucci A. J. , Beyerle G.
Published: September 2003

Analysis of atmospheric occultation data from the GPS Meteorology experiment has revealed that the refractivity retrievals in the lower troposphere were systematically smaller than those obtained with numerical weather prediction models. It has been suggested that the bias was due to a combination of atmospheric multipath, critical refraction, and receiver tracking errors. In this paper, we show that a similar bias exists in the CHAMP and SAC‐C data and describe the characteristics of the bias based on over 6700 soundings from October 2001. Retrievals obtained using the recently introduced canonical transform method are shown to markedly reduce the refractivity bias; however, a significant bias still remains below 2 km altitude. To better understand the underlying causes of the bias, we perform an end‐to‐end simulation study that incorporates full‐wave signal propagation and realistic receiver tracking effects using an ensemble of atmospheric profiles. We find that atmospheric ducting effects associated with the top of the planetary boundary layer (PBL) at 1–2 km altitude would cause retrieval errors at and below the PBL even in the absence of the receiver errors. Furthermore, current implementation of the receiver tracking algorithm based on an enhanced version of the phase‐locked loop could introduce additional errors under the low signal‐to‐noise ratio conditions that are often encountered in the lower troposphere. The latter problem is expected to be resolved in the near future through the adoption of open‐loop tracking and the removal of the navigation modulation from the GPS signal.
Novae on Venus: Geology, classification, and evolution
Authors: Krassilnikov Anton S. , Head James W.
Published: September 2003

Novae are “radially fractured centers” 100–300 km in diameter, and 64 have been identified on Venus. Dense radial fracturing and upraised topography are common, and massive amounts of volcanism are seen in some. Novae are interpreted to be the result of updoming and fracturing of the surface due to interaction of mantle diapirs with the lithosphere and radial fracturing caused by dike emplacement. We analyzed the topography and character of deformational and morphological structures of all 64 novae, leading to a better understanding of their evolution and relation to diapir dynamics, and their age. We subdivided novae into four topographic classes: (1) upraised, (2) annular, (3) flat and negative, and (4) plateau‐like. Novae are located mostly in areas of regional rises and rift zones, with a small number in the lowlands. About one half the novae studied show association with rifts; in some of these the rift troughs surround nova rises and form plateau‐like novae. Plateau‐like novae may predate, postdate, or form simultaneously with rifts. Our analysis generally confirms and updates previous models of novae evolution, with the following geological factors being important: (1) depth of the neutral buoyancy level of the rising mantle diapir; (2) rheological characteristics of the overlying lithosphere; (3) whether or not the visco‐plastic material above the diapir undergoes spreading; and (4) the nature and influence of regional stress and rift structures. These combined factors exert control on the direction of nova evolution and appear to determine the primary characteristics of nova, such as topography, tectonics, and volcanism. We found clear evidence for (1) the important role of dike swarm emplacement in the formation of radial patterns of novae and (2) the formation of concentric tectonic features during the relaxation stage of novae. Novae have multiple evolutionary stages and are long‐lived structures. Stratigraphic analysis of all 64 local areas showed a similarity in the sequence of regional geologic units. We found that 40.3% of the novae population started to form before emplacement of regional plains with wrinkle ridges and that 11.3% completed their activity before this time; 88.7% of the population of novae was active after regional plains formation. In contrast to novae, coronae activity was greatest before formation of regional plains, which may be due to thickening of the lithosphere with time. Detailed structural analysis shows that novae evolution does not always lead to the formation of corona‐like features.
Estimating the benefits of maintaining adequate lake levels to homeowners using the hedonic property method
Authors: Loomis John , Feldman Marvin
Published: September 2003

The hedonic property method was used to estimate residents' economic benefits from maintaining high and stable lake levels at Lake Almanor, California. Nearly a thousand property transactions over a 14‐year period from 1987 to 2001 were analyzed. The linear hedonic property regression explained more than 60% of the variation in‐house prices. Property prices were negatively and significantly related to the number of linear feet of exposed lake shoreline. Each additional one foot of exposed shoreline reduces the property price by $108–$119. A view of the lake added nearly $31,000 to house prices, while lakefront properties sold for $209,000 more than non‐lake front properties.
Properties of high heliolatitude solar energetic particle events and constraints on models of acceleration and propagation
Authors: Dalla S. , Balogh A. , Krucker S. , Posner A. , Müller‐Mellin R. , Anglin J. D. , Hofer M. Y. , Marsden R. G. , Sanderson T. R. , Tranquille C. , Heber B. , Zhang M. , McKibben R. B.
Published: September 2003

We analyse 9 large solar energetic particle (SEP) events detected by the Ulysses spacecraft at high heliolatitudes during the recent solar maximum polar passes. Properties of time intensity profiles from the Ulysses/COSPIN instrument are compared with those measured by SOHO/COSTEP and Wind/3DP near Earth. We find that onset times and times to maximum at high latitude are delayed compared to in‐ecliptic values. We show that the parameter which best orders these characteristics of time profiles is the difference in latitude between the associated flare and the spacecraft. We find that the presence of a shock is not necessary for the establishing of near equal intensities at Ulysses and in the ecliptic during the decay phase. The model of SEP acceleration by coronal mass ejection driven shocks does not appear to account for our observations, which would more easily be explained by particle diffusion across the interplanetary magnetic field.
Short‐term water dynamics in Chihuahua City, Mexico
Authors: Fullerton Thomas M. , Nava Ana Cecilia
Published: September 2003

Time series analysis of water consumption patterns has not been investigated on a wide‐scale basis. For many municipalities such efforts offer a means for developing potentially useful planning tools. Because data requirements are not extensive, model development is feasible in most areas of the world. The work at hand examines the applicability of such a tool in Chihuahua City, an important metropolitan economy in northern Mexico. Sample data are from January 1988 through December 2000. In addition to estimating a linear transfer function equation of water consumption in this city, the model is subjected to a series of simulation benchmark tests.
The use of daclizumab as induction therapy in combination with tacrolimus and mycophenolate mofetil in recipients with previous transplants
Authors: Ciancio Gaetano , Mattiazzi Adela , Roth David , Kupin Warren , Miller Joshua , Burke George W.
Published: September 2003

Abstract: Clinical trials using daclizumab as induction therapy in combination with tacrolimus (TAC) and mycophenolate mofetil (MMF) have been shown to reduce the incidence of acute rejection episodes in solid organ transplantation. In an attempt to obtain a low rejection rate we proceeded with the use of daclizumab as induction therapy, in combination with TAC and MMF for recipients with previous transplants. In this study, we analyzed patients who received previous transplants, treated with the above immunosuppressive regimen. Group A consisted of four patients with previous liver transplants, group B consisted of 16 recipients with previous kidney transplants and group C consisted of three patients with previous simultaneous pancreas–kidney transplants. All patients underwent cadaveric kidney transplants except one patient in group B, who underwent a pancreas transplant. At 12 months, patient and graft survival for all groups was 100 and 100%, respectively. Acute rejection rate was 0% for group A, 12.5% for group B, and 0% for group C. Daclizumab induction therapy is effective for patients with previous transplants and does not appear to increase the risk of acute rejection.
Impairment of the blood‐brain barrier can result in tacrolimus‐induced reversible leucoencephalopathy following heart transplantation
Authors: Kaczmarek Ingo , Groetzner Jan , Meiser Bruno , Mueller Markus , Landwehr Peter , Ueberfuhr Peter , Bruning Roland , Reichart Bruno
Published: September 2003

Abstract:  Fatal leucoencephalopathy is a rare calcineurin inhibitor‐related complication, especially in kidney and liver transplant recipients. The only means of clinical management reported so far is the discontinuation or reduction in the calcineurin inhibitor. We herein report a case of a 37‐yr‐old male who developed leucoencephalopathy 12 wk after heart transplantation and recovered after stabilization of metabolism and arterial blood pressure. The findings in this case support the hypothesis that tacrolimus‐associated neurotoxicity is severely increased by an impairment of the blood‐brain barrier. Withdrawal of tacrolimus was not necessary while other causes of endothelial injury were treated successfully.
Transfusion‐associated graft vs. host disease after donor‐specific leukocyte transfusion before kidney transplantation
Authors: van der Mast Barbara J , van den Dorpel Marinus A , Drabbels Jos JM , Claas Frans HJ , Weimar Willem
Published: September 2003

Transfusion‐associated graft vs. host disease (TA‐GVHD) is a well‐known but rare complication that follows infusion of histo‐incompatible lymphoid cells, often seen in individuals with impaired cellular immunity. However, we present here a case report of fatal TA‐GVHD in a ‘presumed’ immunocompetent patient after transfusion of a freshly isolated buffycoat from a relative as part of our protocol to prepare the patient for living‐related kidney transplantation. To confirm the diagnosis of TA‐GVHD, a polymerase chain reaction was used to detect donor cells in various affected tissues. Furthermore, the immune reactivity of the patient against donor and vice versa was tested on samples taken before transfusion using limiting dilution assays. Our patient received a transfusion with blood from a donor who was homozygous at the human leukocyte antigen (HLA) class I loci. Despite incompatibility for HLA class II, infused donor T lymphocytes were not rejected and became engrafted. The patient did not have cytotoxic T lymphocytes to reject the donor cells. DNA polymorphism studies on several organ biopsies confirmed the presence of infiltrating cells of donor origin. This report illustrates the possibility, in the general patient population, of developing TA‐GVHD from whole blood transfusion. In the case of pre‐transplant blood transfusion, the patient and donor have to be HLA‐typed and special care should be taken in the situation of donor homozygosity for HLA class I, even in the presence of HLA class II incompatibility. Protocols in which donor‐specific blood or bone marrow transfusions are given in an attempt to modulate the immune system should exclude these combinations.
Antiphospholipid syndrome in Asians: clinical manifestations, serological markers and outcome of the National University of Singapore/National University Hospital antiphospholipid cohort
Authors: Yoon Kam Hon , Fong Kok Yong , Sivalingam P. , Koh D. R. , Ng S. C. , Lim T. C. , Kassim S.
Published: September 2003

Background: There is a paucity of studies on antiphospholipid syndrome (APS) in Asian patients.

Aim:  This is the first study in Singapore to describe the clinical features, serological markers and outcomes of one of the largest cohort of APS patients in South‐east Asia.

Method: One hundred and forty‐six patients were studied.

Results:  Within the study group, 89 were primary APS and 57 secondary APS. Age range: 16–84 years (mean Age: 50.1 years). Male : female ratio: 1 : 2.1. Racial distribution: Chinese, 69.2%; Malays, 18.5%; Indians, 11.7%. Systemic lupus erythematosus was the commonest disease associated in 48 patients (32.9%), while 17 (11.6%) had lupus‐like disease. Eighty‐two (56.2%) patients had arterial thrombosis, 37 (25.3%) patients had venous thrombosis, 14 (9.6%) had arterial and venous thrombosis while 17 (11.6%) had obstetric manifestations; 28.8% had thrombocytopenia. Clinical manifestations were diverse with neurological (49.3%), cardiac (40%), renal (23.6%) involvement. There were also several unusual presentations. Recurrent thrombosis occurred in 24.6%. Two had catastrophic APS and there were 12 deaths (9.6%). Antiphospholipid antibody testing included lupus anticoagulant, serum aCL IgG, IgM and IgA, anti‐β2 glycoprotein I (aβ2‐GPI) IgG, IgM, and IgA. The serological positivity rates of the various antiphospholipid antibodies were reported. aβ2‐GPI was positive in 67% of those tested, of which aβ2‐GPI IgA was the most common subtype. The presence of the aβ2‐GPI was associated with recurrent venous thrombosis.

Conclusion:  The clinical manifestations of APS in Asian patients were diverse and multisystemic. Recurrent thrombosis rate was high with significant morbidity and mortality.
Screening for the RA‐associated shared epitope by flow cytometry: comparison of two methods with high resolution genotyping
Authors: Williamson A. A. , Varney M. , Tait B. , Wicks I. P.
Published: September 2003

Objective: To assess flow cytometry‐based detection of the shared rheumatoid epitope (SE) as a potential screening test for high resolution genotyping in patients with rheumatoid arthritis.

Methods:  Peripheral blood mononuclear cells or white blood cell preparations from 84 patients with RA (1987 ACR criteria) were stained with a monoclonal antibody to the SE and analyzed by flow cytometry. The need for additional staining of T‐cells was also assessed. Results with both approaches were compared to high resolution genotyping.

Results:  The test had a positive predictive value of 100%, however, the negative predictive value was 77% due to poor staining in HLA‐DRB1*0101 or *0102 and HLA‐DRB1*1001 positive samples. Although there were no false positive tests with the simplified staining method, the negative predictive value was 61% with poor staining in HLA‐DRB1*0101 and failure to detect 4/22 samples with the high risk allele HLA‐DRB1*0401.

Conclusions:  Without further modification, this approach cannot be recommended as a screening test for high resolution genotyping.
Antiphospholipid (Hughes) syndrome: a review with update
Authors: Malaviya Anand N.
Published: September 2003

This article reviews the evolution of the concept of Hughes syndrome starting with the discovery of beef heart extract‐reactive antibodies in sera of patients with syphilis, presence of such antibodies in some normal individuals who went on to develop systemic lupus erythematosus (SLE), identification of the antigen as anionic phospholipids and some of the cofactors of the blood coagulation cascade, development of more refined techniques for detecting such antibodies and finally its implication in the causation of an acquired hypercoagulable state. The review then discusses the results of extensive international collaborative efforts of the major centres with interest and expertise in Hughes syndrome. It summarizes the recently enunciated classification criteria for this disease. The results and the implications of some of the recent seminal papers, especially on the frequency of different clinical manifestations in a large cohort of patients followed over long‐periods, and recommendations for long‐term high‐intensity anticoagulation for the optimum management of this disease, are also discussed.
Current management of cartilage defects: a review
Authors: Hui James Hoi Po , Marchie Anthony
Published: September 2003

Cartilage defects are a significant cause of morbidity and diminished quality of life, often leading to osteoarthritis (OA). Pain relief, restoration of joint function, and prevention or delay of the onset of OA are the aims of management, and initial treatment is usually conservative but temporary. Recent advances in understanding cartilage biology and the use of diagnostic imaging techniques have, however, allowed more aggressive approaches to cartilage repair which include: arthroscopic lavage with or without debridement; marrow stimulation procedures; implantation of autologous cells or tissue; and knee reconstruction. The role of these techniques in the clinician's effort to match treatment to a patient's needs and expectations is reviewed.
Outcome measures in rheumatoid arthritis
Authors: Malaviya Anand N.
Published: September 2003

Assessing outcome in acute illness and chronic conditions with well defined end‐points, is simple. However, assessing outcome in slowly progressive chronic diseases with natural fluctuations in clinical course is a major challenge. Most chronic musculoskeletal diseases fall in the latter category with rheumatoid arthritis (RA) as its prototype. To make matters worse, there are several aspects of musculoskeletal disease that need to be assessed over time. Degree of synovial inflammation, extent of structural damage, the degree of functional derangement, all need to be assessed separately at intervals. Any intervention must favourably affect each of these parameters to be meaningful to the patient. Having to deal with this problem in routine clinical practise as well as clinical research, rheumatologists have been pioneers in the development and use of clinical measures for outcome assessment in chronic fluctuating illnesses.

 This review summarizes the advances in the field of outcome assessment for RA with emphasis on decreased synovitis. For clinical research the use of Paulus statistics and ACR improvement criteria using a core‐set of parameters is recommended. For day‐to‐day assessment in the clinic DAS28 pooled‐index is recommended. Use of DAS28 is easy and quick, yet it gives internationally acceptable objective assessment of disease activity that compares well with ACR criteria. Availability of DAS28 Calculator v1.1‐beta on‐line at web site www.das‐score.nl or http://www.das‐score.nl/www.das‐score.nl/DAS28‐ne.xls. that performs all the calculations automatically makes it even easier to use in a busy clinic.
An introduction to pharmacoeconomic evaluation in rheumatology
Authors: Chuen LI Shu
Published: September 2003

Pharmacoeconomics is a relatively new healthcare discipline that borrows from several disciplines including health economics, decision analysis and evidence‐based medicine. By using various techniques such as cost‐minimization analysis (CMA), cost‐effectiveness analysis (CEA), cost‐utility analysis (CUA) and cost‐benefit analysis (CBA), pharmacoeconomic evaluation attempts to instil the element of cost‐effectiveness into the clinical decision‐making process beyond the traditional approach of considering efficacy and safety.

 The ultimate aim of pharmacoeconomics is to assist the decision makers in achieving allocation efficiency in the healthcare system. This is in response to the increased need for accountability during the age of cost‐containment in healthcare. The rapidly increasing healthcare costs and the ageing of populations in the developed countries have contributed significantly to the need for consideration of effectiveness in the evaluation of any healthcare treatment, particularly in pharmaceuticals. The growth of this field has proceeded rapidly in the past decade as health policy makers have faced a continuing series of decisions about funding new clinical therapies in an era of increasingly constrained healthcare resources. However, economics of healthcare includes not just an assessment of the cost of a new therapy, but an assessment of its overall economic and clinical impact. The discipline of pharmacoeconomics is developed to fulfil this role.

 To date, the most successful applications of the principles of pharmacoeconomics are in the areas of purchase decision (e.g. decision for formulary inclusion), subsidy decision (e.g. level of subsidy for pharmaceuticals), establishing practise standards (e.g. development of clinical practise guidelines) and assessment of technologies (e.g. medical procedures and technologies).

 The proper application of pharmacoeconomic evaluation in clinical medicine would necessitate a multidisciplinary approach requiring expertise and inputs from practitioners from clinical medicine, biostatistics and psychometrics, among others. A properly conducted pharmacoeconomic evaluation would provide valuable insights for clinicians to allow the choosing of options representing the best value‐for‐money without compromising the quality of care delivered.

 The application of pharmacoeconomic principles in the practise of rheumatology will be illustrated using the example of a hypothetical example of evaluating an application for formulary inclusion of a COX‐2 inhibitor.
Effects of white clover cultivar and companion grass on winter survival of seedlings in autumn‐sown swards
Authors: Elgersma A. , Schlepers H.
Published: September 2003

The aim was to study the effects of white clover cultivar and combinations with perennial ryegrass cultivars on seedling establishment in autumn‐sown swards and on winter survival of seedlings. Large‐leaved white clover cv. Alice and small‐leaved white clover cv. Gwenda, and an erect and a prostrate perennial ryegrass cultivar were sown in autumn in pure stands and as four binary grass‐clover mixtures. Mixtures of white clover cv. Huia and Aberherald with perennial ryegrass were also sown. Companion grasses had no significant impact on the establishment of white clover. The number of seedlings of white clover cv. Alice in mixtures (335 m−2) was higher than cv. Gwenda (183 m−2) and pure swards had similar white clover population densities as mixed swards. White clover cv. Huia tended to have more seedlings than Aberherald (355 and 205 m−2 respectively). No stolons were produced prior to a severe winter, because of the late sowing date. Winter survival of clover seedlings was 0·56 in mixtures and 0·69 in pure stands, irrespective of white clover or companion grass cultivar. Stolon development of white clover in autumn is often considered essential for overwintering survival and spring growth. In this study, there was considerable survival of the non‐stoloniferous tap‐rooted seedlings of all four clover cultivars despite a severe winter.
How do severity and frequency of grazing affect sward characteristics and the choices of sheep during the grazing season?
Authors: Garcia F. , Carrère P. , Soussana J. F. , Baumont R.
Published: September 2003

The effect of grazing frequency and severity on sward characteristics and preferences by sheep was investigated from April to September. Two levels of grazing severity were imposed by varying the numbers of ewes grazing 200 m2 plots for 24 h: four (S, severe) or two (L, lax) ewes. Grazing frequency was either 1 d week−1 (F, frequent) or 1 d every 2 weeks (I, infrequent). By combining frequency and severity, four treatments were obtained: SF, LF, SI and LI. The six binary combinations (SF/LF, SF/SI, SF/LI, LF/SI, LF/LI and SI/LI) were studied in preference tests.

Treatments LF, SI and LI were characterized by a high sward surface height, biomass and amount of reproductive green tissues relative to treatment SF. Herbage quality was not different between the grazing treatments between April and July. In September, after a 6‐week period of regrowth, herbage quality was significantly higher for the SF treatment than the other treatments.

The sheep preferred the swards grazed at a low frequency between April and July, and then changed their preference in favour of the sward with higher quality herbage (treatment SF). The relative abundance of green laminae and the relative digestibility of the swards helped to explain the preferences observed. For a low grazing pressure at the spatio‐temporal scale studied, sheep should graze swards at a relatively low frequency but at a high severity of grazing rather than the reverse.
Somaclonal breeding of reed canarygrass (Phalaris arundinacea L.)
Authors: Gyulai G. , Mester Z. , Kiss J. , Szeman L. , Idnurm A. , Heszky L.
Published: September 2003

Tissue culture‐induced variants (somaclones) of reed canarygrass (Phalaris arundinacea L.) were developed from callus cells of aseptic spikelet cultures. Molecular polymorphism of the populations of somaclones and spikelet‐donor plants as natural vegetative clones were compared by the application of polymerase chain reaction (PCR)‐based techniques of random amplified polymorphic DNA (RAPD)‐, simple sequence repeat (SSR)‐, and inter‐simple sequence repeat (ISSR)‐PCR to genomic DNA samples. The genetic diversity was higher in the somaclones compared with vegetative clones as measured by the maximum genetic distance (MaxGD) which increased from 0·401 (vegetative clones) to 0·498 (somaclones). Along with this change, the minimum genetic distance (MinGD) decreased from 0·20 (control plants) to 0·06 (somaclones) which demonstrated that genetic changes occurred not only in a positive (dissimilarity, MaxGD), but also in a negative (similarity, MinGD) direction. Structural carbohydrate analyses were also performed on the leaves to compare the somaclones with the donor vegetative clones. The mean neutral‐detergent and acid‐detergent fibre concentrations of the leaves of the somaclones were about 0·20 lower than the donor vegetative clones suggesting that increases in the digestibility could be obtained from the somaclonal material.
CAG repeat length in the androgen receptor gene affects the risk of male infertility
Authors: Asatiani K. , von Eckardstein S. , Simoni M. , Gromoll J. , Nieschlag E.
Published: September 2003

During recent years several studies have suggested that a slight increase in the number of CAG repeat sequences in exon 1 of the androgen receptor gene causes idiopathic oligozoospermia. We tested whether CAG repeats are more numerous in men with idiopathic infertility compared to those with known causes of oligozoospermia. CAG repeats were analysed in a consecutive sample of 217 infertile men covering a wide range of diagnoses and sperm counts. Data were compared with those of a control group of 131 normozoospermic men including 62 fathers. CAG repeats (x ± SD) did not differ between idiopathically (21.4 ± 2.9) and non‐idiopathically infertile men (21.6 ± 2.8) or normozoospermic men of unproven fertility (20.6 ± 3.0). Only fathers had significantly fewer repeats (19.4 ± 3.1; p < 0.001). Different from controls, no correlation between CAG repeats and any semen parameter existed in patients. Comparison of our and published studies showed that odds ratios for infertility in men with CAG repeat length in the upper quartile of the normal range increased when the controls were selected by proven fertility. We conclude that more numerous CAG repeats do not directly cause oligozoospermia and propose that men with longer CAG repeats might be more prone to develop infertility in response to any pathogen/epigenetic factors.
Deficit in cytochrome c oxidase activity induced in rat sperm mitochondria by in vivo exposure to zidovudine
Authors: Roger Dominique Leandri , Emmanuel Dulioust , Eric Benbrik , Pierre Jouannet , Marta De Almeida
Published: September 2003

A decreased sperm motility has been reported in men treated with nucleoside analog reverse transcriptase inhibitors (NRTI). Sperm motility is correlated with enzymatic activities of the sperm mitochondrial respiratory chain (MRC), which may be impaired by NRTI. We compared sperm and skeletal muscle MRC and citrate synthase (CS) activities, sperm adenosine triphosphate (ATP) content and sperm motility between rats exposed to zidovudine (AZT) for 10 weeks and controls. Decreased levels of CS‐normalized cytochrome c oxidase (COX, the MRC complex IV) activity were observed in the spermatozoa from AZT‐treated rats, with no significant decrease in ATP content or motility. In muscle absolute COX activity increased after exposure to AZT but CS‐normalized COX activity remained unchanged. These results suggest that exposure to NRTI can induce MRC dysfunction earlier in spermatozoa than in skeletal muscle.
On the mechanism of α‐amylase
Authors: Oudjeriouat Naïma , Moreau Yann , Santimone Marius , Svensson Birte , Marchis‐Mouren Guy , Desseaux Véronique
Published: September 2003

Two inhibitors, acarbose and cyclodextrins (CD), were used to investigate the active site structure and function of barley α‐amylase isozymes, AMY1 and AMY2. The hydrolysis of DP 4900‐amylose, reduced (r) DP18‐maltodextrin and maltoheptaose (catalysed by AMY1 and AMY2) was followed in the absence and in the presence of inhibitor. Without inhibitor, the highest activity was obtained with amylose, kcat/Km decreased 103‐fold using rDP18‐maltodextrin and 105 to 106‐fold using maltoheptaose as substrate. Acarbose is an uncompetitive inhibitor with inhibition constant (L1i) for amylose and maltodextrin in the micromolar range. Acarbose did not bind to the active site of the enzyme, but to a secondary site to give an abortive ESI complex. Only AMY2 has a second secondary binding site corresponding to an ESI2 complex. In contrast, acarbose is a mixed noncompetitive inhibitor of maltoheptaose hydrolysis. Consequently, in the presence of this oligosaccharide substrate, acarbose bound both to the active site and to a secondary binding site. α‐CD inhibited the AMY1 and AMY2 catalysed hydrolysis of amylose, but was a very weak inhibitor compared to acarbose.β‐ and γ‐CD are not inhibitors. These results are different from those obtained previously with PPA. However in AMY1, as already shown for amylases of animal and bacterial origin, in addition to the active site, one secondary carbohydrate binding site (s1) was necessary for activity whereas two secondary sites (s1 and s2) were required for the AMY2 activity. The first secondary site in both AMY1 and AMY2 was only functional when substrate was bound in the active site. This appears to be a general feature of the α‐amylase family.
Arsenite stimulated glucose transport in 3T3‐L1 adipocytes involves both Glut4 translocation and p38 MAPK activity
Authors: Bazuine Merlijn , Ouwens D. Margriet , Gomes de Mesquita Daan S. , Maassen J. Antonie
Published: September 2003

The protein‐modifying agent arsenite stimulates glucose uptake in 3T3‐L1 adipocytes. In the current study we have analysed the signalling pathways that contribute to this response. By subcellular fractionation we observed that arsenite, like insulin, induces translocation of the GLUT1 and GLUT4 glucose transporters from the low‐density membrane fraction to the plasma membrane. Arsenite did not activate early steps of the insulin receptor (IR)‐signalling pathway and the response was insensitive to inhibition of phosphatidylinositol‐3′‐kinase (PI‐3′) kinase by wortmannin. These findings indicate that the ‘classical’ IR–IR substrate–PI‐3′ kinase pathway, that is essential for insulin‐induced GLUT4 translocation, is not activated by arsenite. However, arsenite‐treatment did induce tyrosine‐phosphorylation of c‐Cbl. Furthermore, treatment of the cells with the tyrosine kinase inhibitor, tyrphostin A25, abolished arsenite‐induced glucose uptake, suggesting that the induction of a tyrosine kinase by arsenite is essential for glucose uptake. Both arsenite and insulin‐induced glucose uptake were inhibited partially by the p38 MAP kinase inhibitor, SB203580. This compound had no effect on the magnitude of translocation of glucose transporters indicating that the level of glucose transport is determined by additional factors. Arsenite‐ and insulin‐induced glucose uptake responded in a remarkably similar dose‐dependent fashion to a range of pharmacological‐ and peptide‐inhibitors for atypical PKC‐λ, a downstream target of PI‐3′ kinase signalling in insulin‐induced glucose uptake. These data show that in 3T3‐L1 adipocytes both arsenite‐ and insulin‐induced signalling pathways project towards a similar cellular response, namely GLUT1 and GLUT4 translocation and glucose uptake. This response to arsenite is not functionally linked to early steps of the IR–IRS–PI‐3′ kinase pathway, but does coincide with c‐Cbl phosphorylation, basal levels of PKC‐λ activity and p38 MAPK activation.
Protein farnesyltransferase inhibitors interfere with farnesyl diphosphate binding by rubber transferase
Authors: Mau Christopher J. D. , Garneau Sylvie , Scholte Andrew A. , Van Fleet Jennifer E. , Vederas John C. , Cornish Katrina
Published: September 2003

Rubber transferase, a cis‐prenyltransferase, catalyzes the addition of thousands of isopentenyl diphosphate (IPP) molecules to an allylic diphosphate initiator, such as farnesyl diphosphate (FPP, 1), in the presence of a divalent metal cofactor. In an effort to characterize the catalytic site of rubber transferase, the effects of two types of protein farnesyltransferase inhibitors, several chaetomellic acid A analogs (2, 4–7) and α‐hydroxyfarnesylphosphonic acid (3), on the ability of rubber transferase to add IPP to the allylic diphosphate initiator were determined. Both types of compounds inhibited the activity of rubber transferases from Hevea brasiliensis and Parthenium argentatum, but there were species–specific differences in the inhibition of rubber transferases by these compounds. Several shorter analogs of chaetomellic acid A did not inhibit rubber transferase activity, even though the analogs contained chemical features that are present in an elongating rubber molecule. These results indicate that the initiator‐binding site in rubber transferase shares similar features to FPP binding sites in other enzymes.
Secretion of macrophage urokinase plasminogen activator is dependent on proteoglycans
Authors: Pejler Gunnar , Winberg Jan‐Olof , Vuong Tram T. , Henningsson Frida , Uhlin‐Hansen Lars , Kimata Koji , Kolset Svein O.
Published: September 2003

The importance of proteoglycans for secretion of proteolytic enzymes was studied in the murine macrophage cell line J774. Untreated or 4β‐phorbol 12‐myristate 13‐acetate (PMA)‐stimulated macrophages were treated with hexyl‐β‐d‐thioxyloside to interfere with the attachment of glycosaminoglycan chains to their respective protein cores. Activation of the J774 macrophages with PMA resulted in increased secretion of trypsin‐like serine proteinase activity. This activity was completely inhibited by plasminogen activator inhibitor 1 and by amiloride, identifying the activity as urokinase plasminogen activator (uPA). Treatment of both the unstimulated or PMA‐stimulated macrophages with xyloside resulted in decreased uPA activity and Western blotting analysis revealed an almost complete absence of secreted uPA protein after xyloside treatment of either control‐ or PMA‐treated cells. Zymography analyses with gels containing both gelatin and plasminogen confirmed these findings. The xyloside treatment did not reduce the mRNA levels for uPA, indicating that the effect was at the post‐translational level. Treatment of the macrophages with xylosides did also reduce the levels of secreted matrix metalloproteinase 9. Taken together, these findings indicate a role for proteoglycans in the secretion of uPA and MMP‐9.
Calcium‐induced activation and truncation of promatrix metalloproteinase‐9 linked to the core protein of chondroitin sulfate proteoglycans
Authors: Winberg Jan‐Olof , Berg Eli , Kolset Svein O , Uhlin‐Hansen Lars
Published: September 2003

In the leukemic macrophage cell‐line THP‐1, a fraction of the secreted matrix metalloproteinase 9 (MMP‐9) is linked to the core protein of chondroitin sulfate proteoglycans (CSPG). Unlike the monomeric and homodimeric forms of MMP‐9, the addition of exogenous CaCl2 to the proMMP‐9/CSPG complex resulted in an active gelatinase due to the induction of an autocatalytic removal of the N‐terminal prodomain. In addition, the MMP‐9 was released from the CSPG through a process that appeared to be a stepwise truncation of both the CSPG core protein and a part of the C‐terminal domain of the gelatinase. The calcium‐induced activation and truncation of the MMP‐9/CSPG complex was independent of the concentration of the complex, inhibited by the MMP inhibitors EDTA, 1,10‐phenanthroline and TIMP‐1, but not by general inhibitors of serine, thiol and acid proteinases. This indicated that the activation and truncation process was not due to a bimolecular reaction, but more likely an intramolecular reaction. The negatively charged chondroitin sulfate chains in the proteoglycan were not involved in this process. Other metal‐containing compounds like amino‐phenylmercuric acetate (APMA), NaCl, ZnCl2 and MgCl2 were not able to induce activation and truncation of the proMMP‐9 in this heterodimer. On the contrary, APMA inhibited the calcium‐induced process, whereas high concentrations of either MgCl2 or NaCl had no effect. Our results indicate that the interaction between the MMP‐9 and the core protein of the CSPG was the causal factor in the calcium‐induced activation and truncation of the gelatinase, and that this process was not due to a general electrostatic effect.
Purification, characterization and molecular cloning of tyrosinase from the cephalopod mollusk, Illex argentinus
Authors: Naraoka Tetsushi , Uchisawa Hidemitsu , Mori Haruhide , Matsue Hajime , Chiba Seiya , Kimura Atsuo
Published: September 2003

Tyrosinase (monophenol, l‐DOPA:oxygen oxidoreductase) was isolated from the ink of the squid, Illex argentinus. Squid tyrosinase, termed ST94, was found to occur as a covalently linked homodimeric protein with a molecular mass of 140.2 kDa containing two copper atoms per a subunit. The tyrosinase activity of ST94 was enhanced by proteolysis with trypsin to form a protein, termed ST94t, with a molecular mass of 127.6 kDa. The amino acid sequence of the subunit was deduced from N‐terminal amino acid sequencing and cDNA cloning, indicating that the subunit of ST94 is synthesized as a premature protein with 625 amino acid residues and an 18‐residue signal sequence region is eliminated to form the mature subunit comprised of 607 amino acid residues with a deduced molecular mass of 68 993 Da. ST94 was revealed to contain two putative copper‐binding sites per a subunit, that showed sequence similarities with those of hemocyanins from mollusks, tyrosinases from microorganisms and vertebrates and the hypothetical tyrosinase‐related protein of Caenorhabditis elegans. The squid tyrosinase was shown to catalyze the oxidation of monophenols as well as o‐diphenols and to exhibit temperature‐dependency of o‐diphenolase activity like a psychrophilic enzyme.
Cyclic genotyping strategies. II: True and perceived utilities under incorrect allele frequency assumptions
Authors: Macrossan P. E. , Kinghorn B. P.
Published: September 2003

This paper investigates the potential problems associated with assuming incorrect population frequencies for segregation analysis when animals are genotyped one by one in a cyclic fashion with segregation analysis carried out at each cycle. The base population allele frequencies of 0.1 and 0.5 studied, with the incorrect frequencies assumed for segregation analysis of 0.5 and 0.1 respectively, were investigated on the basis of their covering the range of possible frequencies and the most erroneous assumption of frequencies. An index modelled using linear regression (LR) was employed to choose the next animal to be genotyped in each cycle, based on segregation analysis at the incorrect frequency. The resultant utility was evaluated both at the correct frequency giving actual utility and at the incorrect frequency, the perceived utility. The results are compared with those in which all segregation analysis was carried out at the correct base population allele frequency. The assumption of incorrect population frequency for segregation analysis leads to a decline in the predictive performances of both indices in terms of the true utility of prediction. When utility is also computed at the incorrect frequency to give perceived utility, the assumption of incorrect population frequency of q = 0.5 for segregation analysis of populations simulated at q = 0.1 leads to a perceived decline in the predictive performance of the LR index. However, the assumption of incorrect population frequency of q = 0.1 for segregation analysis of populations simulated at q = 0.5 leads to a perceived increased utility. This latter paradox is explained in terms of genotype probabilities with reference to the method of construction of the genotype probability index.
Cyclic genotyping strategies. I: A comparison of ranking criteria
Authors: Macrossan P. E. , Kinghorn B. P.
Published: September 2003

This research compares three different indices for ranking animals for genotyping for a single biallelic locus. The indices were designed to predict which animal to genotype in each genotyping cycle in order to maximize the utility of the resulting information across the entire population. In each genotyping cycle, a single animal is genotyped, segregation analysis used to provide genotype probabilities for the loci of ungenotyped animals, and the index applied to determine which animal to genotype in the next cycle. The first index is based on a linear regression model combining seven herd and individual animal attributes, with the remaining two indices based on genotype probability index (GPI) and numerator relationship (CON). The linear model gives superior predictive performance, in terms of utility (herd average GPI) for the initial 5% of the population genotyped. After this point, the best index is (CON−GPI), which combines a high degree of relationship of the animal to all other live animals in the pedigree, with a low GPI.
Structural characterization of the porcine pyruvate carboxylase (PC) gene
Authors: Dávalos G. , Sànchez A. , Vidal O. , Noguera J. L. , Amills M.
Published: September 2003

We have characterized the near complete coding sequence of the pig pyruvate carboxylase (PC) gene in six individuals belonging to the Iberian, Large White and Piétrain breeds. The pig PC nucleotide sequence was 3.517 bp long and encoded one 1.171 amino acid protein displaying a remarkable level of amino acid identity (75–92%) with other vertebrate pyruvate carboxylase sequences. The pig PC mRNA was expressed in several tissues such as liver, backfat, abdominal fat, ovary, uterus and testicle. Sequence aligment allowed to identify one silent (C/T)837 polymorphism at exon 6 that was typed in a number of Piétrain, Landrace and Large White pigs by using a primer extension‐based approach. Allelic frequencies were 0.23 (Piétrain), 0.64 (Landrace) and 0.47 (Large White) for the C allele. Radiation hybrid mapping of the pig PC locus revealed that this gene maps to 2p17, near the SW2623 and the SW256 genetic markers.
Single nucleotide polymorphism genotyping of bovine milk protein genes using the tetra‐primer ARMS‐PCR
Authors: Rincón G. , Medrano J. F.
Published: September 2003

We demonstrate the use and optimization of the tetra‐primer amplification refractory mutation system‐polymerase chain reaction (tetra‐primer ARMS‐PCR) as an efficient technique to genotype single nucleotide polymorphism (SNP) of milk protein genes. The technique utilizes a set of four primers in a single PCR reaction tube without the need of post‐PCR manipulations to accomplish an efficient classification. The technique was successfully applied to genotype SNP in the bovine κ‐casein, β‐casein, αS1‐casein and α‐lactalbumin genes.