bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoprecipitation: Denaturing Lysis
Authors: Harlow Ed , Lane David
Published: September 2006

INTRODUCTION

For many sources of antigens, one useful method of lysis is to treat cells with harsh, denaturing solutions to release most of the protein antigens, as described here. The lysates are then diluted to reduce the denaturing conditions to levels that are suitable for the formation of antibody-antigen complexes. The resulting solution is precleared prior to immunoprecipitation. The major consideration in choosing this lysis method is that all noncovalent protein-protein interactions will be lost and that denaturation-sensitive epitopes will be destroyed. Therefore, this is not an appropriate method for many types of final assays, but it is a good method for quantitation of the antigen, careful identification of the polypeptide band recognized by the antibody, and display of denaturation-resistant epitopes.
Immunoprecipitation: Preclearing the Lysate
Authors: Harlow Ed , Lane David
Published: September 2006

INTRODUCTION

To reduce backgrounds and to improve the signal-to-noise ratio, an antibody that does not recognize the antigen being studied can be added to the lysate and processed as for a normal immunoprecipitation. Any nonspecific proteins that might contaminate the final immunoprecipitation step are presumably removed with this irrelevant antibody. Rabbit serum collected from a healthy adult animal that has not been previously immunized is used because it provides a complex mixture of different antibody types. Fixed Staphylococcus aureus Cowan I (SAC), a relatively inexpensive, concentrated source of protein A is used to collect the rabbit antibodies. Precleared samples prepared according to this protocol are ready for the final immunoprecipitation step.
SDS-Polyacrylamide Gel Electrophoresis of Proteins
Authors: Sambrook Joseph , Russell David W.
Published: September 2006

INTRODUCTION

This protocol describes the separation of proteins by SDS-polyacrylamide gel electrophoresis. SDS is used with a reducing agent and heat to dissociate the proteins. SDS-polypeptide complexes form and migrate through the gels according to the size of the polypeptide. By using markers of known molecular weight, the molecular weight of the polypeptide chain(s) can be estimated.
Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array: Cloning by Homologous Recombination and High-Throughput Transformation
Authors: Hazbun Tony R. , Miller John P.
Published: September 2006

INTRODUCTION

Although described here as a means of producing baits and preys, the generation of an array of ORFs fused to any moiety can be achieved using this protocol. This procedure capitalizes on the asexual/sexual reproductive cycle of yeast cells. Laboratory yeast strains can be maintained indefinitely by asexual reproduction as one of two haploid mating types, MATa or MATα. However, overlaying MATa and MATα cells allows them to fuse and form diploid cells. Hence, the strategy involves transforming MATα cells with the Gal4 BD (bait) fusion plasmids and transforming MATa cells with the Gal4 AD (prey) fusion plasmids. The transformants are then mixed on a plate, or in liquid, and diploids are selected by plating them onto media lacking the marker nutrients for both of the plasmids. In this way, the prey transformations need only be performed once, and each bait protein can then be tested against the array of prey-containing strains using only one transformation per bait. The protocol is easily adapted to small-scale transformation of single microcentrifuge tubes for bait transformations.
Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array: Storage, Maintenance, and Working with Living Arrays
Authors: Hazbun Tony R. , Miller John P.
Published: September 2006

INTRODUCTION

The most difficult aspect of using an array approach for a high-throughput study is maintaining the array’s viability while avoiding contamination of the plates. This protocol describes three different storage methods for the array. Frozen stocks are the most permanent method and are an absolute requirement for maintaining a record of the array. Unfortunately, because thawing is necessary to access the strains from a 96-well format, the repeated use of the frozen stocks as a source is expected to eventually lead to loss of viability of the stock. (Generating frozen stocks of the array strains individually, as well as in the arrayed format, is essential.) Maintenance of a water stock provides a source that is more convenient and (relative to yeast cells in media) semi-permanent (~6 months). However, the water stock is fairly precarious because cross-transfer of strains between wells can occur quite easily if the plates are dropped or disturbed. The third method of storage, that of maintaining extra copies of the solid medium array plates, is the most convenient, but it is also the least stable. In short, the best option is to make several frozen stocks. For greater convenience in repeated use of the array, generate water stocks and additional copies of the solid medium plates.
Hop stunt viroid is processed and translocated in transgenic Nicotiana benthamiana plants
Authors: GÓMEZ GUSTAVO , PALLÁS VICENTE
Published: September 2006

Viroids are small, single‐stranded, circular, pathogenic RNAs that do not code for proteins and thus depend on host factors for their replication and movement. They induce diseases in plants by direct interaction with host factors through a mechanism as yet unidentified. Hop stunt viroid (HSVd) has been found in a wide range of herbaceous and woody hosts. Nicotiana benthamiana is one of the most frequently used experimental systems in the study of plant–virus interactions. However, this plant is a non‐host for HSVd. To go further in the study of the requirements of a plant to be a host for viroids N. benthamiana was transformed with cDNA expressing dimeric (+) HSVd. Correct processing to the circular (+) monomers was always observed, demonstrating that N. benthamiana has the appropriate machinery to cleave and circularize (+) HSVd molecules. Additionally, N. benthamiana plants agroinoculated with the dimeric (–) HSVd accumulated the circular and linear (+) monomers, indicating that (–) HSVd transcripts can be used as template for the RNA–RNA transcription. Grafting assays showed that HSVd is able to move to distal plant parts. In addition, alterations in the normal flower development were observed. Taken together, these results indicate that deficiencies in the interaction with host factors related either to the early steps of the replication process or to cell to cell movement appear to be the factors that limit infectivity of HSVd in N. benthamiana. These transgenic plants can be an useful tool to study the HSVd–host factor interactions.
Plant species as indicators of the extent of desertification in four sandy rangelands
Authors: An Ping , Inanaga Shinobu , Zhu Nanwen , Li Xiangjun , Fadul Hassan M. , Mars Messaoud
Published: September 2006

We examined the distribution of plants in sandy rangelands with different degrees of desertification in (i) Horqin and Mu Us sandy lands in north China, (ii) Darfur district in north‐west Sudan and (iii) southern Tunisia. The severity of desertification in these areas was classified as light, moderate, severe or extreme. The following vegetation changes were observed with increasing severity of desertification: a decrease in the number of plant species, an increase in drought‐tolerant plants, a decrease in the proportion of palatable grasses and the occurrence of some species in only one category of desertification, whereas other species occurred in several categories. Plant species with unique niche requirements were identified and recommended as indicators of the degree of desertification for particular areas. We discuss the characteristics of species in areas with severe desertification and the method of using plant species as indicators of the extent of desertification.
Oxidative stress and inflammation in iron‐overloaded patients with β‐thalassaemia or sickle cell disease
Authors: Walter Patrick B. , Fung Ellen B. , Killilea David W. , Jiang Qing , Hudes Mark , Madden Jacqueline , Porter John , Evans Patricia , Vichinsky Elliott , Harmatz Paul
Published: September 2006

Blood transfusion therapy is life‐saving for patients with β‐thalassaemia and sickle cell disease (SCD), but often results in severe iron overload. This pilot study examined whether the biomarkers of tissue injury or inflammation differ in these two diseases. Plasma malondialdehyde (MDA) was significantly increased 1·8‐fold in thalassaemia relative to control patients. In contrast, MDA in SCD was not significantly different from controls. In multivariate analysis, the strongest predictors of elevated MDA were liver iron concentration (P < 0·001) and specific diagnosis (P = 0·019). A significant 2‐fold elevation of non‐transferrin bound iron (NTBI) was observed in thalassaemia relative to SCD. NTBI was not a significant predictor of high MDA in multivariate analysis. SCD patients showed a significant 2·2‐fold elevation of the inflammatory marker interleukin (IL)‐6 relative to controls, and a 3·6‐ and 1·7‐fold increase in IL‐5 and IL‐10 relative to thalassaemia. Although α‐tocopherol was significantly decreased by at least 32% in both thalassaemia and SCD, indicating ongoing oxidant stress and antioxidant consumption, γ‐tocopherol, a nitric oxide‐selective antioxidant, was increased 36% in SCD relative to thalassaemia. These results demonstrate that thalassaemia patients have increased MDA and circulating NTBI relative to SCD patients and lower levels of some cytokines and γ‐tocopherol. This supports the hypothesis that the biology of SCD may show increased inflammation and increased levels of protective antioxidants compared with thalassaemia.
Outcome in patients with Wiskott–Aldrich syndrome following stem cell transplantation: an analysis of 57 patients in Japan
Authors: Kobayashi Ryoji , Ariga Tadashi , Nonoyama Shigeaki , Kanegane Hirokazu , Tsuchiya Shigeru , Morio Tomohiro , Yabe Hiromasa , Nagatoshi Yoshihisa , Kawa Keisei , Tabuchi Ken , Tsuchida Masahiro , Miyawaki Toshio , Kato Shunichi
Published: September 2006

A total of 57 patients with Wiskott–Aldrich syndrome (WAS) were studied after undergoing stem cell transplantation (SCT) in Japan between January 1985 and December 2004. Eleven patients received transplants from human leucocyte antigen (HLA)‐matched related donors, 10 from HLA‐mismatched related donors, 21 from unrelated bone marrow donors, and 15 from unrelated cord blood donors. Nine of the 57 patients rejected the initial graft. The overall 5‐year survival rate was 73·7% and the 5‐year failure‐free survival rate was 65·7% (failure was defined as rejection or death). The overall 5‐year survival rates for patients receiving bone marrow and cord blood from unrelated donors were both 80·0%. Based on univariate analysis, the factors associated with poor survival were: transplantation from an HLA‐mismatched related donor, patient age of more than 5 years at the time of transplantation, and a conditioning regimen other than busulfan and cyclophosphamide (BU‐CY) or busulfan, cyclophosphamide and antithymocyte globulin (BU‐CY‐ATG). In a multivariate analysis, a conditioning regimen other than BU‐CY and BU‐CY‐ATG was the only independent factor associated with transplantation failure. Given the improved outcome for WAS patients following transplantation from an unrelated donor, we conclude that patients with WAS should receive SCT as soon as possible after diagnosis.
Cytology is useful in breast screening: results and long‐term follow‐up of the Singapore Breast Screening Pilot Project
Authors: Chew I. , Tan Y. , Tan P. H.
Published: September 2006

Objective: The Singapore Breast Screening Pilot Project (SBSPP) was embarked upon (1994–1997) to determine if mammography was useful in early breast cancer detection among Asian women.

Patients and measurements: Of 28 231 women screened, fine needle aspiration cytology (FNAC) was performed in 232 individuals as part of the triple assessment.

Results: Absolute and complete sensitivities for the diagnosis of carcinoma were 46.7% and 82.2%, respectively, based on the results of FNAC. Specificity was 63.3%. The inadequate rate was 31%. Five women who were considered cancer‐free on triple assessment and, in two cases open diagnostic biopsy during the SBSPP, subsequently developed breast cancer after a median follow‐up of 6 years.

Conclusion: Although our FNAC results compared relatively well with international standards, they reflect a small cohort, and may face additional difficulties in a larger programme.
Mastopathic fibroadenoma of the breast: a pitfall of aspiration cytology
Authors: Mori I. , Han B. , Wang X. , Taniguchi E. , Nakamura M. , Nakamura Y. , Bai Y. , Kakudo K.
Published: September 2006

Objective:  To assess the correlation between cytological diagnoses and histological subtypes of fibroadenoma (FA) and to clarify the cytological features of a specific group of FA displaying variable features similar to fibrocystic disease (mastopathic type, MFA), and to evaluate the significance of this subtype in cytological diagnosis.

Methods:  A review of 141 cases of histologically proven FA was performed. We re‐classified them into four subtypes according to Kinoshita's criteria [Jpn J Breast Cancer6 (1991) 377] and further selected 92 cases for which both fine needle aspiration (FNA) smears and histological specimens were available. Among them, 18 cases of MFA and their cytological smears were selected for the present study.

Results:  There was significant correlation between MFA and cytological diagnosis of ‘indeterminate’ or ‘suspicious for malignancy’ (P < 0.01). Although no false‐positive diagnosis was experienced in our series, 56% of the MFAs (10/18) had cytological diagnoses of indeterminate or were included in the category ‘suspicious for malignancy’. Smears from MFA revealed high cellularity (9/18 smears had more than 10 epithelial clusters each composed of more than 50 cells), presence of cellular discohesiveness (13/18, 72.2%), but only mild nuclear atypia (5/18, 27.8%). Anisonucleosis was present in fewer than half the cases and no apparent condensed chromatin was identified.

Conclusion:  We highlight the significance of subclassification of MFA in aspiration cytology of breast. MFA had a significantly higher chance of falling into the ‘suspicious for malignancy’ or ‘indeterminate’ diagnostic category in aspiration cytology. It might be a diagnostic challenge for cytopathologist to identify this subtype of FA in FNA smears.
Body size changes in ground beetle assemblages – a reanalysis of Braunet al. (2004)’s data
Authors: LÖVEI GÁBOR L. , MAGURA TIBOR
Published: September 2006

Abstract1. Data inBraunet al.’s (2004)recent paper on size trends in ground beetles (Carabidae) are re‐analysed, after re‐classifying the species according to feeding categories.

2. When ground beetles are classified as predatory species, mixed feeders or herbivores, and evaluated separately, the size trends indicate group‐specific differences. Most of these do not support the efficiency‐specialisation hypothesis.

3. Taxonomic groups rarely fit into one ecological category and ecological analyses should explicitly consider this hurdle.
The origin of the savanna biome
Authors: BEERLING DAVID J. , OSBORNE COLIN P.
Published: September 2006

Savannas are a major terrestrial biome, comprising of grasses with the C4 photosynthetic pathway and trees with the C3 type. This mixed grass–tree biome rapidly appeared on the ecological stage 8 million years ago with the near‐synchronous expansion of C4 grasses around the world. We propose a new hypothesis for this global event based on a systems analysis that integrates recent advances in how fire influences cloud microphysics, climate and savanna ecology in a low carbon dioxide (CO2) world. We show that fire accelerates forest loss and C4 grassland expansion through multiple positive feedback loops that each promote drought and more fire. A low CO2 atmosphere amplifies this cycle by limiting tree recruitment, allowing the ingress of C4 grasses to greatly increase ecosystem flammability. Continued intensification of land use could enhance or moderate the network of feedbacks that have initiated, promoted and sustained savannas for millions of years. We suggest these alterations will overprint the effects of anthropogenic atmospheric change in coming decades.
Regulation of surfactant protein and defensin mRNA expression in cultured ovine type II pneumocytes by all‐trans retinoic acid and VEGF
Authors: Grubor B. , Meyerholz D. K. , Lazic T. , DeMacedo M. M. , Derscheid R. J. , Hostetter J. M. , Gallup J. M. , DeMartini J. C. , Ackermann M. R.
Published: September 2006

Beta‐defensins and surfactant proteins are components of the pulmonary innate immune system. Their gene expression is regulated by development, hormones, growth and immunoregulatory factors. It was our hypothesis that growth and differentiation factors such as all‐trans retinoic acid (RA) and vascular endothelial growth factor (VEGF) may affect expression of selected innate immune genes by respiratory epithelial cells. Ovine JS7 cells (alveolar type II pneumocytes) were incubated in serum‐free Dulbecco's modified Eagle's medium (DMEM) complete media that contained: no treatment (negative control), RA (500 nM), or VEGF (100 ng/ml) for 6, 12 or 24 h incubation. Total RNA was isolated, cDNA synthesized, and relative mRNA levels of surfactant protein A (SP‐A) and SP‐D, and sheep beta‐defensin‐1 (SBD‐1) were determined by real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR). Cells had significantly increased expression of SP‐D mRNA at 6 h and 24 h, decreased expression of SP‐A mRNA at 12 h, and unchanged levels of SBD‐1 mRNA after the treatment with RA compared with their respective negative controls. VEGF did not alter the expression of the three innate immune genes. These findings suggest that SP‐A and SP‐D have different transcription regulation pathways, and that expression of SBD‐1 is not inducible by RA similar to its human homolog HBD‐1. The lack of changes induced by VEGF treatment suggests that VEGF does not have a direct effect on epithelial cells, but may affect gene expression indirectly.
Percutaneous endoscopic gastrostomies: the burden of treatment from a patient perspective
Authors: Jordan Sue , Philpin Sue , Warring Joanne , Cheung Wai Yee , Williams John
Published: September 2006

Aim.  This paper reports a study of patients’ experiences of this relatively new technology, and explores possible strategies for defining, delineating and addressing patients’ concerns, problems and needs.

Background.  The number of patients in the community relying on percutaneous endoscopic gastrostomy for their nutritional needs is increasing, but percutaneous endoscopic gastrostomy feeding is not problem‐free. However, few studies report on its impact from a patient perspective.

Methods.  Twenty adults with long‐term percutaneous endoscopic gastrostomies were interviewed in their own homes, using both semi‐structured and structured approaches. Qualitative data were analysed thematically and related to a symptom checklist and an established quality of life measure, the Short Form‐12.

Findings.  Most participants felt that insertion of a percutaneous endoscopic gastrostomy had been life‐saving, but found that percutaneous endoscopic gastrostomy feeding came to dominate their lives and was associated with an appreciable ‘burden of treatment’. Some, but not all, of the problems recounted were unavoidable: percutaneous endoscopic gastrostomy feeding inevitably imposed physical restrictions on mobility; 17/20 participants had experienced serious technical problems with their percutaneous endoscopic gastrostomy tubes. On structured questioning, all interviewees had some, potentially treatable, gastrointestinal symptoms, and eight had continuous problems. In some cases, the burden of treatment was exaggerated by being unanticipated and, for 13 patients, difficulties with care provided by non‐specialist services. Describing this ‘burden of treatment’ for each patient required both a narrative and a structured approach: without the symptom checklists, some important and treatable problems were missed. Both physical and mental health Short Form‐12 scores were low, and mental health scores were congruent with the interview data.

Conclusions.  It might be possible to reduce the ‘burden of treatment’ identified by incorporating a structured approach to patient monitoring and by reviewing the occupational territories of specialist and non‐specialist practitioners. Further research with larger numbers of participants is needed to explore the integration of narrative and quantitative data when determining patients’ clinical needs.
Factors contributing to frailty: literature review
Authors: Levers Merry‐Jo , Estabrooks Carole A. , Ross Kerr Janet C.
Published: September 2006

Aim.  This paper presents a review of theoretical and research literature in order to identify the factors contributing to frailty.

Background.  Frailty is a multifaceted gerontological concept that lacks a clear definition, but may result from an identifiable homogeneous cluster of bio–psycho–social–spiritual factors.

Method.  A total of 134 articles were identified through a search of the MEDLINE (1966 to July 2004), CINAHL (1982 to July 2004), PsychInfo (1985 to July 2004) and Ageline (1995 to July 2004) databases. Each article was reviewed to determine its fit with inclusion/exclusion criteria. Seven research and 11 theoretical articles were retained and further reviewed for methodological quality using a validity tool.

Findings.  Seventeen different definitions of frailty were identified. Regardless of the differing definitions, common contributing factors could be identified. Physical, cognitive/psychological, nutritional and social factors, as well as ageing and disease, were evident in both the theoretical and research literature.

Conclusions.  Although there is strong agreement that a relationship exists between a cluster of factors and frailty, designation of the factors as contributors or outcomes of frailty differs. Without a clear explanatory theory of the path from contributors to frailty to outcomes, research will continue to produce confusing results. A theoretical framework that includes bio–psycho–social–spiritual factors as contributors to frailty is recommended as the most useful framework for gerontological nursing.
Developing nursing and midwifery research capacity in a university department: case study
Authors: Green Barbara , Segrott Jeremy , Hewitt Jeanette
Published: September 2006

Aim.  This paper reports a case study which examined the selection, implementation and outcomes of one university department's approach to building research capacity in nursing and midwifery.

Background.  The literature identifies building nursing research capacity as an important challenge. In countries such as Australia and the United Kingdom it is taking place in the context of the move of nurse education into the university sector. Structural and cultural barriers to building academic nursing research capacity exist. Previous studies highlight the strategies that academic departments adopt to build nursing research capacity.

Methods.  Using case study methodology, data were collected using documentary analysis and semi‐structured interviews with a purposive sample of 27 academic and related staff and a focus group with seven staff. The data were collected between 2003 and 2005.

Findings.  The department had adopted an inclusive approach to capacity development (allowing all teaching staff to develop their research capability), but is now moving towards a more focused path and the cultivation of ‘leading edge’ research. Neophyte researchers described lacking confidence in undertaking research, and expressed a need for more formal support structures. The importance of effective management of capacity building was highlighted, including transparent communication and mapping of existing capacity. Key external influences included the lack of core research funding, and divergence between the university's emphasis on research and the department's desire to develop an interface between teaching, research and clinical practice.

Conclusion.  Academic leadership and educational management should work in tandem. Staff development and the provision of time and support are crucial aspects of research capacity development. The effectiveness of ‘inclusive’ and ‘exclusive’ approaches to research capacity development depends on the nature of each department, and they are not mutually exclusive.
Care of patients with chronic obstructive pulmonary disease in primary health care
Authors: Lundh Lena , Rosenhall Leif , Törnkvist Lena
Published: September 2006

Aim.  This paper is a report of a study of nurses’ perceptions of caring for patients with chronic obstructive pulmonary disease.

Background.  Chronic obstructive pulmonary disease is a global health problem and the number of patients being treated with this disease in primary healthcare settings is increasing. This places new demands on the nurses involved.

Method.  A phenomenographic approach was adopted, using a purposive sample. Data were generated between February and May 2003 from 20 interviews with district nurses and general nurses who cared for patients in primary healthcare settings with chronic obstructive pulmonary disease.

Findings.  In most cases, nurses cared for older people with moderate to severe chronic obstructive pulmonary disease. They described this care from two overall orientations: task and individual. The nurses’ perceptions of the care of these patients were described as creating commitment and participation by establishing a good relationship with patients and supporting them in their personal care, educating patients by supplying information and knowledge in various ways, co‐operation by co‐operating with or referring to other caregivers, and arranging and implementing clinical examinations and treatments.

Conclusion.  The type of care depended on who the patient met: either a task‐oriented nurse or an individual‐oriented nurse. Therefore, nursing programmes should pay special attention to the support and guidance of new and inexperienced and task‐oriented nurses. Healthcare planners should take into consideration the need for individualized care when organizing care and allocating resources for chronically ill people.
Validation and reproducibility of a semi‐quantitative Food Frequency Questionnaire for use in elderly Swiss women
Authors: Dumartheray E. Wynn , Krieg M‐A. , Cornuz J. , Whittamore D. R. , Lovell D. P. , Burckhardt P. , Lanham‐New S. A.
Published: September 2006

Objective  The principal aim of this study was to develop a Swiss Food Frequency Questionnaire (FFQ) for the elderly population for use in a study to investigate the influence of nutritional factors on bone health. The secondary aim was to assess its validity and both short‐term and long‐term reproducibility.

Design  A 4‐day weighed record (4 d WR) was applied to 51 randomly selected women of a mean age of 80.3 years. Subsequently, a detailed FFQ was developed, cross‐validated against a further 44 4‐d WR, and the short‐ (1 month, n = 15) and long‐term (12 months, n = 14) reproducibility examined.

Setting  French speaking part of Switzerland.

Subjects  The subjects were randomly selected women recruited from the Swiss Evaluation of the Methods of Measurement of Osteoporotic Fracture cohort study.

Results  Mean energy intakes by 4‐d WR and FFQ showed no significant difference [1564.9 kcal (SD 351.1); 1641.3 kcal (SD 523.2) respectively]. Mean crude nutrient intakes were also similar (with nonsignifcant P‐values examining the differences in intake) and ranged from 0.13 (potassium) to 0.48 (magnesium). Similar results were found in the reproducibility studies.

Conclusion  These findings provide evidence that this FFQ adequately estimates nutrient intakes and can be used to rank individuals within distributions of intake in specific populations.
C‐reactive protein, heart rate variability and prognosis in community subjects with no apparent heart disease
Authors: SAJADIEH A. , NIELSEN O. W. , RASMUSSEN V. , HEIN H. O. , HANSEN J. F.
Published: September 2006

Objectives.  Increased C‐reactive protein (CRP) and reduced heart rate variability (HRV) both indicate poor prognosis. An inverse association between HRV and CRP has been reported, suggesting an interaction between inflammatory and autonomic systems. However, the prognostic impact of this interaction has not been studied. We thus investigated the prognostic impact of CRP, HRV and their combinations.

Design.  Population‐based study.

Subjects.  A total of 638 middle‐aged and elderly subjects with no apparent heart disease from community.

Methods.  All were studied by clinical and laboratory examinations, and 24‐h Holter monitoring. Four time domain measures of HRV were studied. All were prospectively followed for up to 5 years.

Results.  Mean age was 64 years (55–75). During the follow‐up, 46 total deaths and 11 cases of definite acute myocardial infarction were observed. Both CRP and three of four HRV measures were significantly associated with increased rate of death or myocardial infarction. In a Cox model with CRP ≥2.5 μg mL−1, standard deviation for the mean value of the time between normal complexes ≤100 ms, and their combination, hazard ratio and 95% CI for subjects with both abnormalities was 3.20 (1.55–6.56), P = 0.0016, and for subjects with either abnormality 1.63(0.83–3.20), P = 0.15, after adjustment for conventional risk factors. The combination of CRP and other measures of HRV gave similar results. This indicates an interaction between CRP and HRV with a synergistic effect.

Conclusions.  The combination of CRP and HRV or heart rate (HR) predicts death and myocardial infarction with synergism, indicating interaction between inflammatory and autonomic systems with a prognostic significance.
Critical review on the withdrawal period calculation for injection site residues
Authors: SANQUER A. , WACKOWIEZ G. , HAVRILECK B.
Published: September 2006

This review concerns a statistical method for calculating withdrawal period for injection site residues. A recently adopted Committee for Medicinal Products for Veterinary Use/European Agency for the Evaluation of Medicinal Product (CVMP/EMEA) guideline recommends to apply the same method for the calculation of withdrawal period for injection site residues as for other edible tissues. For reasons in this study developed below, this approach is deemed to be inappropriate for the injection site residues. The injection site residues often violate regression assumptions with regard to homoscedasticity (same variance in residue concentrations for different slaughter times) and linearity (of the mean depletion curve in loge‐scale). The currently recommended method cannot adequately handle these aspects. An alternative pragmatic method taking into account the last slaughter time with all data below the reference threshold, combined with a safety span, is proposed for injection site residues. A nonparametric approach for calculating the withdrawal period is also presumed to be a sound alternative. The references commonly used are the Maximum Residue Limit (MRL) and the Acceptable Daily Intake (ADI). Unfortunately these references are not relevant to the acute risk exposure associated with injection site consumption. The use of alternative references, such as the Acceptable Single Dose Intake (ASDI) or the Acute Reference Dose (ARD) are thought to be more appropriate.
The Parkes Multibeam Pulsar Survey – VI. Discovery and timing of 142 pulsars and a Galactic population analysis
Authors: Lorimer D. R. , Faulkner A. J. , Lyne A. G. , Manchester R. N. , Kramer M. , McLaughlin M. A. , Hobbs G. , Possenti A. , Stairs I. H. , Camilo F. , Burgay M. , D'Amico N. , Corongiu A. , Crawford F.
Published: September 2006

We present the discovery and follow-up observations of 142 pulsars found in the Parkes 20-cm multibeam pulsar survey of the Galactic plane. These new discoveries bring the total number of pulsars found by the survey to 742. In addition to tabulating spin and astrometric parameters, along with pulse width and flux density information, we present orbital characteristics for 13 binary pulsars which form part of the new sample. Combining these results from another recent Parkes multibeam survey at high Galactic latitudes, we have a sample of 1008 normal pulsars which we use to carry out a determination of their Galactic distribution and birth rate. We infer a total Galactic population of 30 000 ± 1100 potentially detectable pulsars (i.e. those beaming towards us) having 1.4-GHz luminosities above 0.1 mJy kpc2. Adopting the Tauris & Manchester beaming model, this translates to a total of 155 000 ± 6000 active radio pulsars in the Galaxy above this luminosity limit. Using a pulsar current analysis, we derive the birth rate of this population to be 1.4 ± 0.2 pulsars per century. An important conclusion from our work is that the inferred radial density function of pulsars depends strongly on the assumed distribution of free electrons in the Galaxy. As a result, any analyses using the most recent electron model of Cordes & Lazio predict a dearth of pulsars in the inner Galaxy. We show that this model can also bias the inferred pulsar scaleheight with respect to the Galactic plane. Combining our results with other Parkes multibeam surveys we find that the population is best described by an exponential distribution with a scaleheight of 330 pc. Surveys underway at Parkes and Arecibo are expected to improve the knowledge of the radial distribution outside the solar circle, and to discover several hundred new pulsars in the inner Galaxy.
Flow cytometric quantification of antigen D sites on red blood cells of partial D and weak D variants in India
Authors: Kulkarni S. , Mohanty D. , Gupte S. , Vasantha K. , Joshi S.
Published: September 2006

summary The D antigenic sites are diminished in weak D and partial D variants. The aim of this study was to estimate D antigen on RBC in weak D and partial D variants in Indian population by using flow cytometric method.

Blood samples of 42 cases of partial D, eight cases of weak D and 123 normal Rh phenotypes identified by serological methods were used in the study. An indirect immunofluorescence method was employed using 29 monoclonal anti‐D as primary antibody. The D antigenic sites were calculated using control RBC as internal standard.

The D antigenic sites in weak D and partial D variants are less than that found in normal Rh phenotypes. In weak D, the D antigenic sites were between 1500 and 7000 D antigens per cell. Among partial D variants, DVI had minimum and DVa had maximum number of D sites.

Flow cytometry is a very good tool for demonstrating minor variation in D antigen when serological methods are inconclusive. The D antigenic sites per RBC in partial D variants identified in Indian population are reported for the first time.
Comparison of the performance of microtube column systems and solid‐phase systems and the tube low‐ionic‐strength solution additive indirect antiglobulin test in the detection of red cell alloantibodies
Authors: Weisbach V. , Kohnhäuser T. , Zimmermann R. , Ringwald J. , Strasser E. , Zingsem J. , Eckstein R.
Published: September 2006

summary To compare the performance of seven currently available test systems in the detection of erythrocyte alloantibodies (ab), we tested in parallel 446 sera samples containing red cell ab [368 sera samples with ab that are assumed to be clinically significant (cs‐ab) and 78 sera samples with ab that are assumed to be of minor clinical significance (ms‐ab)] using the tube spin low‐ionic‐strength solution (addition method) indirect antiglobulin test (tube LISS‐IAT), three microtube column agglutination techniques (DiaMed‐ID, Ortho BioVue and Bio‐Rad Scangel), one affinity adherence test system (CLB/Mast CellBind Screen) and two solid‐phase tests [Biotest Solidscreen II and Immucor Capture‐R Ready‐Screen (4)]. To address the specificity of the three test systems under routine conditions, results of 4566 patient samples obtained using the tube LISS‐IAT, results of 5205 patient samples obtained using the Scangel and results of 3560 samples obtained using the Capture‐R were evaluated. The DiaMed‐ID detected 344 cs‐ab and 43 ms‐ab, BioVue 333 cs‐ab and 48 ms‐ab, Scangel 348 cs‐ab and 62 ms‐ab, CellBind Screen 346 cs‐ab and 47 ms‐ab, Solidscreen 330 cs‐ab and 38 ms‐ab, Capture‐R 358 cs‐ab and 45 ms‐ab and LISS‐IAT 159 cs‐ab and 12 ms‐ab. In routine practice, erythrocyte cs‐ab could be identified in 61 (67·8%) of 90 reactive sera (specificity: 98·6%) in the tube LISS‐IAT, in 169 (58·7%) of 288 (94·4%) in Bio‐Rad Scangel and in 101 (51·0%) of 198 reactive sera (94·3%) in Capture‐R. We conclude that the sensitivity of the microcolumn, affinity adherence and solid‐phase test systems in the detection of cs‐ab was similar and was markedly superior to that of the conventional tube LISS‐IAT. All high‐sensitive test systems produced higher rates of false positives and ms‐ab compared to the tube test. An individual cost–benefit analysis, considering the recent knowledge about the clinical significance of weak‐reactive cs‐ab, should be performed in every institution to decide whether and if so which high‐sensitive screening system should be applied.
SI25 The Capillary Haemoglobin Level is a More Physiological Measure than the Venous Level and is Less Susceptible to Seasonal Fluctuation When the Donor Haemoglobin Level is Close to the Regulatory Cut‐Off
Authors: Darragh E. , McSweeney E. , Ekin C. , Kinsella A. , Murphy W. G.
Published: September 2006

Introduction We set out to show that at levels of venous haemoglobin in blood donors that were close to the regulatory limits of 13.5 g dL−1 for males and 12.5 g dL−1 for females, capillary haemoglobin levels of 0.5 g less (13.0 g dL−1 for males, 12.0 g dL−1 for females) were biologically equivalent; that is, setting a capillary haemoglobin cut‐off for donations at 13.0/12.0 g dL−1 is in compliance with a regulatory requirement of 13.5/12.5 g dL−1 for venous haemoglobin levels. At the same time we compared seasonal variations in venous haemoglobin levels with capillary haemoglobin levels at levels close to the regulatory cut‐off points.

Methods Male and female donors with capillary haemoglobin levels of 13.0–13.4 for males and 12.0–12.4 g dL−1 for females as measured by HemoCue (Angelholm, Sweden) were accepted for donation, and a venous haemoglobin level using the CellDyn 4000 instrument (Abbott Diagnostics, Mount View, CA, USA) was measured on the routine EDTA sample taken at donation from the diversion pouch. All donations meeting these criteria were compared for the two haemoglobin levels over a period of one calendar year (May 2004–April 2005). Differences were tested for statistical significance using Pearson coefficient, Student's t‐test, and confidence interval analysis.

Results A total of 11, 991 donations were analysed (8341 females, 3650 males). Mean capillary haemoglobin levels were 13.1 (SD 0.2) g dL−1 for males, and 12.1 (SD 0.2) g dL−1 for females, as expected for the conditions. Mean monthly capillary haemoglobin levels varied between 13.0–13.2 (SD 0.2) g dL−1 for males, and 12.0–12.2 (SD 0.2) g dL−1 for females. Mean venous haemoglobin levels were 14.1 (SD 0.7) for males and 12.8 (SD 0.6) for females. Mean monthly venous haemoglobin levels showed a wider month‐to‐month variation, from 13.8 to 14.3 for males and 12.5 to 12.9 for females. The monthly variation for August was significantly different from other months for both males and females, and the venous haemoglobin in this population was significantly correlated with average monthly temperature (P = 0.01).

Conclusions (i) A cut‐off for capillary haemoglobin of 12.0 g dL−1 for females and 13.0 g dL−1 for males equates to a venous haemoglobin level that meets regulatory requirements, and is less sensitive to seasonal fluctuations than venous haemoglobin levels; and (ii) Venous haemoglobin levels fluctuate in healthy populations at the expense of maintaining capillary haemoglobin levels, consistent with the presence of a physiological mechanism to preserve steady‐state tissue oxygenation in healthy humans in the presence of modest temperature fluctuations. This requires further study, especially in relation to determining optimum capillary haemoglobin levels in stressed states.
P39 The Second Example of LU:‐4: a Serological and Molecular Study
Authors: Karamatic Crew V. , Warke N. , Ahrens N. , Poole J. , Daniels G.
Published: September 2006

Introduction  The Lutheran (LU) blood group system consists of 19 antigens numbered 1–21 (LU10, LU15 designated obsolete). There are four pairs of allelic and polymorphic antigens; Lua/Lub(LU1/LU2), LU6/LU9, LU8/LU14 and Aua/Aub(LU18/LU19). The remaining 11 are all antigens of high frequency. For the majority of Lutheran antigens the molecular bases have been determined and their expression is nearly always associated with a single amino acid change in the Lu‐glycoprotein. Lutheran antigens are carried on two red cell membrane glycoproteins, Lu‐glycoprotein and epithelial cancer antigen. The two isoforms are encoded by a single gene LU located on chromosome 19q13.2 and organised into 15 exons.

Case Study and Methods  We investigated a case of a prenatal patient with no known history of transfusion or previous pregnancies, whose serum contained an antibody giving positive reactions with all donor cells tested. Serological tests were performed by standard agglutination techniques. Genomic DNA was isolated from whole blood of the patient, all the exons of the LU gene were amplified by PCR and directly sequenced.

Results  Serological tests confirmed the presence of a Lutheran antibody in the patient's serum. All tested cells were incompatible with the patient's serum, except several examples of Lu(a‐b‐) cells of both the dominant and recessive phenotype. Her red cells were tested with antibodies to high frequency Lutheran antigens and failed to react with anti‐Lu4. Sequencing of the LU gene revealed a novel homozygous mutation in exon 5 524G>T, causing an amino acid change Arg175Leu in the protein. This novel mutation was found in the same codon (523CGG) in LU exon 5 as the only other known example of LU:‐4, where the change at the same nucleotide was 524G>A, leading to Arg175Gln substitution.

Conclusion  We report the second individual with the rare LU:‐4 phenotype. We have confirmed the patient's serum contained an anti‐Lu4 antibody. This phenotype results from a single point mutation in LU leading to an Arg175Leu amino acid change in the protein. This new point mutation occurred at the same nucleotide as the mutation in the only other known example of LU:‐4. However, it caused a different amino acid substitution than in the original example, indicating a potential hot spot for mutations in LU.
Seed dormancy and germination in the summer annual Galeopsis speciosa
Authors: KARLSSON LM , ERICSSON JAL , MILBERG P
Published: September 2006

This study examined germination and dormancy in Galeopsis speciosa (Lamiaceae), a common summer annual weed in cold‐temperate areas. Seeds collected in southern Sweden were subjected to several experiments. The seeds were dormant at maturity. Seeds sown outdoors after collection produced a small number of seedlings that emerged early in the spring. After long cold stratification or stratification outdoors over two winters, the maximum germination was 40–50%; germination occurring over a wide range of temperatures. Warm stratification preceding cold stratification had no effect on germination, but repeated warm and cold periods seemed to promote germination. Gibberellic acid (GA) stimulated germination, but full germination was only achieved after more than 2 months of incubation at the most suitable temperature regime tested. Excised embryos grew and developed into normal seedlings. With these results, the species does not fit into the currently used system for seed dormancy classifications. The response to GA and the growth of excised embryos indicate non‐deep or intermediate physiological dormancy, but dormancy alleviation by stratification was not in line with the guiding principles for these classifications. Galeopsis speciosa has a strong dormancy that is sufficiently alleviated during the winter to allow germination of only part of a seed batch each year; hence a stepwise germination pattern occurs over a period of several years.
Ranking of invasive woody plant species for management on Réunion Island
Authors: TASSIN J , RIVIÈRE J‐N , CAZANOVE M , BRUZZESE E
Published: September 2006

The native flora of Réunion Island in the Indian Ocean is threatened by invasive woody plants introduced for agriculture, forestry or as ornamentals. We reviewed archives to identify woody plants introduced since colonisation and ranked the biological impact of the main non‐indigenous species on native vegetation. The relationship between cumulative number of non‐indigenous plants and population follows an s‐shaped curve, but the rate of introduction was mainly determined by historical periods with five main introduction phases identified. A total of 318 introduced woody species were recorded with 132 identified as naturalized in natural ecosystems. Of these, 26 of the more serious invasive species (i.e. having a large biological impact on the new environment) were selected and ranked by biological impact to native ecosystems. Hiptage benghalensis, Ligustrum robustum var. walkeri, Acacia mearnsii, Ulex europaeus and Psidium cattleianum were identified as having the highest invasiveness capacity and the greatest impacts on succession and utilization of resources in natural areas. A strategy is required to manage high‐priority invasive species and reduce the rate of non‐indigenous plant introductions to the island.
A target‐site mutation is present in a glyphosate‐resistant Lolium rigidum population
Authors: WAKELIN AM , PRESTON C
Published: September 2006

Annual ryegrass (Lolium rigidum) is the only weed species to have evolved resistance to the broad‐spectrum herbicide glyphosate in Australia. A population that had failed to be controlled by glyphosate was collected from a vineyard in the Adelaide Hills region of South Australia. Dose–response experiments on this population (SLR 77) showed that it was glyphosate resistant, with an LD50 that was 1.9–3.4 times higher than that of a susceptible population (VLR 1). The movement of radiolabelled glyphosate within SLR 77 plants showed that this population did not have the differential glyphosate translocation mechanism of resistance common to several other Australian glyphosate‐resistant populations. Subsequent analysis of shikimic acid accumulation within the plant after glyphosate treatment showed that this population accumulated significantly less shikimic acid than a susceptible population, but more than a glyphosate‐resistant population with the translocation mechanism, indicating the possible involvement of another mechanism of resistance. Sequencing of a portion of the SLR 77 5‐enolpyruvylshikimate‐3‐phosphate synthase gene was carried out and a mutation causing an amino acid change at position 106 from proline to threonine was identified. This mutation is likely to be responsible for glyphosate resistance in this population, as mutations in this position have been found to be responsible for glyphosate resistance in goosegrass (Eleusine indica) from Malaysia. This paper represents the first report of target‐site glyphosate resistance in L. rigidum and provides evidence that this species has at least two mechanisms of glyphosate resistance present in Australia.