Integration of retrotransposons-based markers in a linkage map of barley

A deeper understanding of random markers is important if they are to be employed for a range of objectives. The sequence specific amplified polymorphism (S-SAP) technique is a powerful genetic analysis tool which exploits the high copy number of retrotransposon long terminal repeats (LTRs) in the plant genome. The distribution and inheritance of S-SAP bands in the barley genome was studied using the Steptoe × Morex (S × M) double haploid (DH) population. Six S-SAP primer combinations generated 98 polymorphic bands, and map positions were assigned to all but one band. Eight putative co-dominant loci were detected, representing 16 of the mapped markers. Thus at least 81 of the mapped S-SAP loci were dominant. The markers were distributed along all of the seven chromosomes and a tendency to cluster was observed. The distribution of S-SAP markers over the barley genome concurred with the knowledge of the high copy number of retrotransposons in plants. This experiment has demonstrated the potential for the S-SAP technique to be applied in a range of analyses such as genetic fingerprinting, marker assisted breeding, biodiversity assessment and phylogenetic analyses.

High yield production of a soluble bifidobacterial β-galactosidase (BbgIV) inE. coliDH5α with improved catalytic efficiency for the synthesis of prebiotic galactooligosaccharides

The bifidobacterial β-galactosidase (BbgIV) was produced in E. coli DH5α at 37 and 30 °C in a 5 L bioreactor under varied conditions of dissolved oxygen (dO2) and pH. The yield of soluble BbgIV was significantly (P < 0.05) increased once the dO2 dropped to 0–2% and remained at such low values during the exponential phase. Limited dO2 significantly (P < 0.05) increased the plasmid copy number and decreased the cells growth rate. Consequently, the BbgIV yield increased to its maximum (71–75 mg per g dry cell weight), which represented 20–25% of the total soluble proteins in the cells. In addition, the specific activity and catalytic efficiency of BbgIV were significantly (P < 0.05) enhanced under limited dO2 conditions. This was concomitant with a change in the enzyme secondary structure, suggesting a link between the enzyme structure and function. The knowledge generated from this work is very important for producing BbgIV as a biocatalyst for the development of a cost-effective process for the synthesis of prebiotic galactooligosaccharides from lactose.

Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (Cd) hyperaccumulator Noccaea caerulescens

Zinc (Zn) and cadmium (Cd) hyperaccumulation may have evolved twice in the Brassicaceae, in Arabidopsis halleri and in the Noccaea genus. Tandem gene duplication and deregulated expression of the Zn transporter, HMA4, has previously been linked to Zn/Cd hyperaccumulation in A. halleri. Here, we tested the hypothesis that tandem duplication and deregulation of HMA4 expression also occurs in Noccaea. A Noccaea caerulescens genomic library was generated, containing 36,864 fosmid pCC1FOS (TM) clones with insert sizes similar to 20-40 kbp, and screened with a PCR-generated HMA4 genomic probe. Gene copy number within the genome was estimated through DNA fingerprinting and pooled fosmid pyrosequencing. Gene copy numbers within individual clones was determined by PCR analyses with novel locus specific primers. Entire fosmids were then sequenced individually and reads equivalent to 20-fold coverage were assembled to generate complete whole contigs. Four tandem HMA4 repeats were identified in a contiguous sequence of 101,480 bp based on sequence overlap identities. These were flanked by regions syntenous with up and downstream regions of AtHMA4 in Arabidopsis thaliana. Promoter-reporter beta-glucuronidase (GUS) fusion analysis of a NcHMA4 in A. thaliana revealed deregulated expression in roots and shoots, analogous to AhHMA4 promoters, but distinct from AtHMA4 expression which localised to the root vascular tissue. This remarkable consistency in tandem duplication and deregulated expression of metal transport genes between N. caerulescens and A. halleri, which last shared a common ancestor > 40 mya, provides intriguing evidence that parallel evolutionary pathways may underlie Zn/Cd hyperaccumulation in Brassicaceae.

CLEC-2 expression is maintained on activated platelets and on platelet microparticles

The C-type lectin-like receptor CLEC-2 mediates platelet activation through a hem-immunoreceptor tyrosine-based activation motif (hemITAM). CLEC-2 initiates a Src- and Syk-dependent signaling cascade that is closely related to that of the 2 platelet ITAM receptors: glycoprotein (GP)VI and FcγRIIa. Activation of either of the ITAM receptors induces shedding of GPVI and proteolysis of the ITAM domain in FcγRIIa. In the present study, we generated monoclonal antibodies against human CLEC-2 and used these to measure CLEC-2 expression on resting and stimulated platelets and on other hematopoietic cells. We show that CLEC-2 is restricted to platelets with an average copy number of ∼2000 per cell and that activation of CLEC-2 induces proteolytic cleavage of GPVI and FcγRIIa but not of itself. We further show that CLEC-2 and GPVI are expressed on CD41+ microparticles in megakaryocyte cultures and in platelet-rich plasma, which are predominantly derived from megakaryocytes in healthy donors, whereas microparticles derived from activated platelets only express CLEC-2. Patients with rheumatoid arthritis, an inflammatory disease associated with increased microparticle production, had raised plasma levels of microparticles that expressed CLEC-2 but not GPVI. Thus, CLEC-2, unlike platelet ITAM receptors, is not regulated by proteolysis and can be used to monitor platelet-derived microparticles.

Evidence for long-term regional changes in precipitation on the East Coast Mountains in Mauritius

Global climate change and its impacts are being increasingly studied and precipitation trends are one of the measures of quantifying climate change especially in the tropics. This study uses daily rainfall data to determine if there are changes in the long-term trends in rainfall variability in the East Coast Mountains of Mauritius during the last few decades, and to investigate the factors influencing the trends in the inter-annual to inter-decadal rainfall variability. Statistical modelling has been used to investigate the trends in total seasonal rainfall, the number of rain days and the mean amount of rain per rainy days and the local, regional and large-scale factors that affect them on inter-annual to inter-decadal time scales. The strongest inter-decadal trend was found in the number of rain days for both rainfall seasons, and the other variables were found to have weak or insignificant trends. Both local factors, such as the surrounding sea surface temperatures and large-scale phenomena such as Indian Monsoon and the El Niño Southern Oscillation were found to influence rainfall patterns.

Global observed changes in daily climate extremes of temperature and precipitation

A suite of climate change indices derived from daily temperature and precipitation data, with a primary focus on extreme events, were computed and analyzed. By setting an exact formula for each index and using specially designed software, analyses done in different countries have been combined seamlessly. This has enabled the presentation of the most up-to-date and comprehensive global picture of trends in extreme temperature and precipitation indices using results from a number of workshops held in data-sparse regions and high-quality station data supplied by numerous scientists world wide. Seasonal and annual indices for the period 1951-2003 were gridded. Trends in the gridded fields were computed and tested for statistical significance. Results showed widespread significant changes in temperature extremes associated with warming, especially for those indices derived from daily minimum temperature. Over 70% of the global land area sampled showed a significant decrease in the annual occurrence of cold nights and a significant increase in the annual occurrence of warm nights. Some regions experienced a more than doubling of these indices. This implies a positive shift in the distribution of daily minimum temperature throughout the globe. Daily maximum temperature indices showed similar changes but with smaller magnitudes. Precipitation changes showed a widespread and significant increase, but the changes are much less spatially coherent compared with temperature change. Probability distributions of indices derived from approximately 200 temperature and 600 precipitation stations, with near-complete data for 1901-2003 and covering a very large region of the Northern Hemisphere midlatitudes (and parts of Australia for precipitation) were analyzed for the periods 1901-1950, 1951-1978 and 1979-2003. Results indicate a significant warming throughout the 20th century. Differences in temperature indices distributions are particularly pronounced between the most recent two periods and for those indices related to minimum temperature. An analysis of those indices for which seasonal time series are available shows that these changes occur for all seasons although they are generally least pronounced for September to November. Precipitation indices show a tendency toward wetter conditions throughout the 20th century.

Developmental changes in the germinability, desiccation tolerance, hardseededness, and longevity of individual seeds of Trifolium ambiguum

Background and Aims: Using two parental clones of outcrossing Trifolium ambiguum as a potential model system, we examined how during seed development the maternal parent, number of seeds per pod, seed position within the pod, and pod position within the inflorescence influenced individual seed fresh weight, dry weight, water content, germinability, desiccation tolerance, hardseededness, and subsequent longevity of individual seeds. Methods: Near simultaneous, manual reciprocal crosses were carried out between clonal lines for two experiments. Infructescences were harvested at intervals during seed development. Each individual seed was weighed and then used to determine dry weight or one of the physiological behaviour traits. Key Results: Whilst population mass maturity was reached at 33–36 days after pollination (DAP), seed-to-seed variation in maximum seed dry weight, when it was achieved, and when maturation drying commenced, was considerable. Individual seeds acquired germinability between 14 and 44 DAP, desiccation tolerance between 30 and 40 DAP, and the capability to become hardseeded between 30 and 47 DAP. The time for viability to fall to 50 % (p50) at 60 % relative humidity and 45 °C increased between 36 and 56 DAP, when the seed coats of most individuals had become dark orange, but declined thereafter. Individual seed f. wt at harvest did not correlate with air-dry storage survival period. Analysing survival data for cohorts of seeds reduced the standard deviation of the normal distribution of seed deaths in time, but no sub-population showed complete uniformity of survival period. Conclusions: Variation in individual seed behaviours within a developing population is inherent and inevitable. In this outbreeder, there is significant variation in seed longevity which appears dependent on embryo genotype with little effect of maternal genotype or architectural factors.

Behavioral changes associated with a population density decline in the facultatively social red fox

Understanding the causal mechanisms promoting group formation in carnivores has been widely investigated, particularly how fitness components affect group formation. Population density may affect the relative benefits of natal philopatry versus dispersal. Density effects on individual behavioral strategies have previously been studied through comparisons of different populations, where differences could be confounded by between-site effects. We used a single population of red foxes (Vulpes vulpes) in the city of Bristol, UK, that underwent a natural perturbation in density to compare key changes in 1) group structure, 2) within-group relatedness, 3) mating system, 4) dispersal, and 5) dominance attainment. At high densities (19.6-27.6 adults km(-2)), group sex ratios were equal and included related and unrelated individuals. At low densities (4.0-5.5 adults km(-2)), groups became female biased and were structured around philopatric females. However, levels of within-group relatedness were unchanged. The genetic mating patterns changed with no instances of multiple-paternity litters and a decline in the frequency of extrapair litters of cubs from <= 77% to <= 38%. However, the number of genetically monogynous groups did not differ between periods. Dispersal was male biased at both high and low densities. At high density, most dominant males in the study groups appeared to have gained dominance after dispersing, but natal philopatry was an equally successful strategy at low density; conversely, most dominant females were philopatric individuals at both high and low densities. These results illustrate how density may alter behavioral strategies such as mating patterns and how this, in turn, alters group structure in a single population.

Histone H4 gene expression and morphological changes on shoot apices of strawberry (Fragaria x ananassa Duch.) during floral induction

To investigate flower induction in June-bearing strawberry plants, morphological changes in shoot apices and Historic H4 expression in the central zone during flower initiation were observed. Strawberry plants were placed under flower inducible, short-day conditions (23 degrees C/17 degrees C, 10 h day length) for differing number of days (8, 16, 20, 24 or 32 days) and then these plants were transferred to non-inducible, long-day conditions (25 degrees C/20 degrees C, 14 h day length). The shoot apices of plants placed under short-day conditions for 8 days were flat, similar to shoot apices of plants in the vegetative phase of development, and Histone H4 was not expressed in the central zone during the experimental period. On the other hand, the shoot apices of plants placed under short-day conditions for 16 days remained flat, similar to shoot apices of plants placed under short-day conditions for 8 days, but Histone H4 was expressed in the central zone at the end of the short-day treatment. Morphological changes in the shoot apices of these plants were observed 8 days after the change in day-length. These plants developed differentiated flower organs after they were grown for another 30 days under long-day conditions. These results indicate that changes in the expression pattern of the Histone H4 gene occur before morphological changes during flower induction and that the expression of the gene in the central zone can be used as one of the indicators of the flowering process in strawberries. (c) 2006 Elsevier B.V. All rights reserved.

Hepatozoon griseisciuri in grey squirrels (Sciurus carolinensis): changes of blood leucocyte numbers resulting from infection

Numbers of leucocytes in squirrels with gametocytes of Hepatozoon in their blood (infected) were compared with animals without gametocytes (uninfected). Typical values for leucocytes/mm(3) blood in uninfected squirrels were: leucocytes 5-7 x 10(3), granulocytes 3-4 x 10(3), lymphocytes 2-0 x 10(3) and monocytes 0-3 x 10(3) cells. Infection caused an increase in monocytes, lymphocytes and granulocytes, and there was a significant positive association between parasitaemia level and numbers of both total leucocytes and monocytes. Infected animals had more uninfected monocytes/mm(3) blood than did uninfected animals. The proportions of monocytes were more variable over time in infected animals, but no shift between infected and uninfected status was detected. Transfer of serum from infected squirrels to mice resulted in elevated counts of total blood leucocytes, monocytes and granulocytes, but not of lymphocytes, as compared with controls. Serum from squirrels with high parasitaemias had a more marked effect than serum from squirrels with low parasitaemias. Results indicate an infection - related monocytosis, possibly controlled by cytokines, that increases the number of cells available for invasion by gametocytes, thus enhancing the chances of parasite transmission.

Changes in milk composition and processing properties during the spring flush period

The period, known to UK farmers and processors as the "spring flush", when the cows' diet changes from dry feed to spring pasture, has long been established as a time of change in milk properties and processing characteristics. Although it is believed to be a time when problems in processing are most likely to occur (e.g. milk that does not form clots or forms weak gels during cheesemaking), there is little evidence in the literature of detailed changes in milk composition and their impact on product manufacture. In this study, a range of physicochemical properties were analysed in milk collected from five commercial dairy herds before, during and after the spring flush period of 2006. In particular, total and ionic calcium contents of milk were studied in relation to other parameters including rennet clotting, acid gel properties, heat coagulation, alcohol stability, micelle size and zeta potential. Total divalent cations were significantly reduced from 35.4 to 33.4 mmol.L-1 during the study, while ionic calcium was reduced from 1.48 to 1.40 mmol.L-1 over the same period. Many parameters varied significantly between the sample dates. However, there was no evidence to suggest that any of the milk samples would have been unsuitable for processing - e.g. there were no samples that did not form clots with chymosin within a reasonable time or formed especially weak rennet or acid gels. A number of statistically significant correlations were found within the data, including ionic calcium concentration and pH; rennet clotting time (RCT) and micelle diameter; and RCT and ethanol stability. Overall, while there were clear variations in milk composition and properties over this period, there was no evidence to support the view that serious processing problems are likely during the change from dry feed to spring pasture.

Changes in physical properties of bovine milk from the colostrum period to early lactation

The aim of this study was to analyze individual cows' samples from the colostrum, postcolostrum, and early lactation periods to investigate how milk composition, physical properties, stability, and suitability for processing change throughout this period. Attention was paid to the first week postpartum in which the composition of bovine mammary secretion can change markedly. Properties including pH, titratable acidity, ethanol stability (ES), rennet clotting time, and casein micelle size were analyzed, together with some compositional factors such as fat, total protein, lactose, total and ionic calcium, magnesium, citrate, phosphorus, sodium, and potassium. Total Ca (36.2 mM) and free ionic Ca (2.58 mM), Mg (5.9 mM), P (32.2 mM), and Na (24.1 mM) appeared to be high on d 5 postpartum, having decreased substantially over the first 5 d; they gradually decreased thereafter. The average pH on d 5 was only 6.49, compared with 6.64 at 1 mo postpartum. Stability measurements showed that the average ES on d 5 was 70% and the rennet clotting time was 12.2 min, which were significantly lower than values at later stages. A number of milk properties including ES, pH, protein content, and Ca2+ concentration could be useful for identifying the point of transition from colostrum to the early lactation period. Knowing the composition and physical properties of colostrum and postcolostrum secretions will help establish when such milk is suitable for processing and determine the best use for that milk.

Physiological changes in Campylobacter jejuni on entry into stationary phase

Campylobacter jejuni NCTC 11168 does not exhibit the general increase in cellular stress resistance on entry into stationary phase that is seen in most other bacteria. This is consistent with the lack of global stationary phase regulatory elements in this organism. deduced from an analysis of its genome sequence. We now show that C. jejuni NCTC 11168 does undergo certain changes in stationary phase, of a pattern not previously described. As cells entered stationary phase there was a change in membrane fatty acid composition, principally a decrease in the proportion of unsaturated fatty acids and an increase in the content of cyclopropane and short-chain fatty acids. These changes in membrane composition were accompanied by an increase in the resilience of the cell membrane towards loss of integrity caused by pressure and an increase in cellular pressure resistance. By contrast. there were no major changes in resistance to acid or heat treatment. A similar pattern of changes in stress resistance on entry, into stationary phase was seen in C. jejuni NCTC 11351, the type strain. These changes appear to represent a restricted Physiological response to the conditions existing in stationary phase cultures, in an organism having limited capacity for genetic regulation and adaptation to environment. &COPY; 2004 Elsevier B.V. All rights reserved.

Changes in phenolic composition and antioxidant activity of virgin olive oil during frying

The concentration of hydroxytyrosol (3,4-DHPEA) and its secoiridoid derivatives (3,4-DHPEA-EDA and 3,4-DHPEA-EA) in virgin olive oil decreased rapidly when the oil was repeatedly used for preparing french fries in deep-fat frying operations. At the end of the first frying process (10 min at 180 degreesC), the concentration of the dihydroxyphenol components was reduced to 50-60% of the original value, and after six frying operations only about 10% of the initial components remained. However, tyrosol (p-HPEA) and its derivatives (p-HPEA-EDA and p-HPEA-EA) in the oil were much more stable during 12 frying operations. The reduction in their original concentration was much smaller than that for hydroxytyrosol and its derivatives and showed a roughly linear relationship with the number of frying operations. The antioxidant activity of the phenolic extract measured using the DPPH test rapidly diminished during the first six frying processes, from a total antioxidant activity higher than 740,mumol of Trolox/kg down to less than 250 mumol/kg. On the other hand, the concentration of polar compounds, oxidized triacylglycerol monomers (oxTGs), dimeric TGs, and polymerized TGs rapidly increased from the sixth frying operation onward, when the antioxidant activity of the phenolic extract was very low, and as a consequence the oil was much more susceptible to oxidation. The loss of antioxidant activity in the phenolic fraction due to deep-fat frying was confirmed by the storage oil and oil-in-water emulsions containing added extracts from olive oil used for 12 frying operations.

Preventing drug related morbidity: a process for facilitating changes in practice

Aim: To describe how quantitative data obtained from applying a series of indicators for preventable drug related morbidity (PDRM) in the electronic patient record in English general practice can be used to facilitate changes aimed at helping to improve medicines management. Design: A multidisciplinary discussion forum held at each practice facilitated by a clinical researcher. Subjects and setting: Eight English general practices. Outcome measures: Issues discussed at the multidisciplinary discussion forum and ideas generated by practices for tackling these issues. Progress made by practices after 1, 3, and 6 months. Results: A number of clinical issues were raised by the practices and ideas for moving them forward were discussed. The issues that were easiest and most straightforward to deal with (for example, reviewing specific patient groups) were quickly addressed in most instances. Practices were less likely to have taken steps towards addressing issues at a systems level. Conclusions: Data generated from applying PDRM indicators can be used to facilitate practice-wide discussion on medicines management. Different practices place different priority levels on the issues they wish to pursue. Individual practice "ownership'' of these, together with having a central committed figure at the practice, is key to the success of the process.