Expression of the protease inhibitor ecotin and its co-crystallization with trypsin

We have expressed the serine protease inhibitor ecotin to high levels (greater than 400 mg/l of cell culture) in its natural mileau, the Escherichia coli periplasm, using the endogenous signal peptide and the heterologous tac promoter. After induction, functional, soluble ecotin comprises 15% of total cellular protein. This expression system has facilitated initiation of a crystallographic study to determine the structural basis for inhibition of the pancreatic serine proteases by ecotin. Ecotin was co-crystallized with rat trypsin mutant D102N. Preliminary crystallographic analysis of co-crystals showed that they diffract to at least 2.7 A, and indicate that they belong to the monoclinic space group, P21. The cell constants are a = 52.0 A, b = 93.3 A, c = 160.7 A, and beta = 96 degrees. Four molecules each of trypsin and ecotin are found in the asymmetric unit.     

Distribution of Sulfur within Oilseed Rape Leaves in Response to Sulfur Deficiency during Vegetative Growth

The distribution of S to sulfate, glucosinolates, glutathione, and the insoluble fraction within oilseed rape (Brassica napus L.) leaves of different ages was investigated during vegetative growth. The concentrations of glutathione and glucosinolates increased from the oldest to the youngest leaves, whereas the opposite was observed for SO₄²⁻. The concentration of insoluble S was similar among all of the leaves. At sufficient S supply and in the youngest leaves, 2% of total S was allocated to glutathione, 6% to glucosinolates, 50% to the insoluble fraction, and the remainder accumulated as SO₄²⁻. In the middle and oldest leaves, 70% to 90% of total S accumulated as SO₄²⁻, whereas glutathione and glucosinolates together accounted for less than 1% of S. When the S supply was withdrawn (minus S), the concentrations of all S-containing compounds, particularly SO₄²⁻, decreased in the youngest and middle leaves. Neither glucosinolates nor glutathione were major sources of S during S deficiency. Plants grown on nutrient solution containing minus S and low N were less deficient than plants grown on solution containing minus S and high N. The effect of N was explained by differences in growth rate. The different responses of leaves of different ages to S deficiency have to be taken into account for the development of field diagnostic tests to determine whether plants are S deficient.     

Molecular characterization of a phage-encoded resistance system in Lactococcus lactis

A specific fragment of the genome of Tuc2009, a temperate lactococcal bacteriophage, was shown to contain several open reading frames, whose deduced protein products exhibited similarities to proteins known to be involved in DNA replication and modification. In this way, a putative single-stranded binding protein, replisome organizer protein, topoisomerase I, and a methylase were identified. When the genetic information coding for the putative replisome organizer protein of Tuc2009, Rep2009, was supplied on a high-copy-number plasmid vector, it was shown to confer a phage-encoded resistance (Per) phenotype on its lactococcal host UC509.9. The presence of this recombinant plasmid was shown to cause a marked reduction in Tuc2009 DNA replication, suggesting that the observed phage resistance was due to titration of a factor, or factors, required for Tuc2009 DNA replication. Further experiments delineated the phage resistance-conferring region to a 160-bp fragment rich in direct repeats. Gel retardation experiments, which indicated a protein-DNA interaction between this 160-bp fragment and the Rep2009 protein, were performed. UC509.9 strains harboring plasmids with randomly mutated versions of this fragment were shown to display a variable phage resistance phenotype, depending on the position of the mutations.     

Factors influencing older people's self reported use of dental services in the UK

Objectives: This study was designed to determine the use of dental services and factors associated with their use among the United Kingdoms' older population. Design: A national study involving 1,116 older people (aged 60 or older). Setting: Home Interview s were undertaken exploring the time and reason for last dental visit. In addition, socio-demographic characteristics and proxy oral health measures (self-reported number of teeth and edentulous status) of the respondents were collected. Results: Forty seven percent (528) claimed they visited the dentist within the past year, 10% (116) claimed that the reason for their last visit was because of a dental emergency, 43% (484) were classified as “regular attenders” - having attended the dentist within the past year for a non dental emergency. Bivariate analysis identified that regular dental attendance was associated with age (P<0.01), social class (P<0.01), income level (P<0.01), educational attainment (P<0.01), self-reported number of teeth possessed (P<0.01) and edentulous status (P<0.01). In regression analysis, self reported edentulous status and number of teeth possessed emerged as the most important factors in determining service utilisation. Possessing a full denture was associated with a 6-fold decrease, having accounted for other factors, in the likelihood of attending the dentist within the past year for a non dental-emergency (OR=0.15, CI 0.10,0.21). Conclusion: Less than half of the sample population were “regular dental attenders”, their attendance was associated with a number of socio-demographic and oral health factors. In particular, edentulous state was a major factor associated with their use of services.     

Variation in trophic shift for stable isotope ratios of carbon, nitrogen, and sulfur

Use of stable isotope ratios to trace pathways of organic matter among consumers requires knowledge of the isotopic shift between diet and consumer. Variation in trophic shift among consumers can be substantial. For data from the published literature and supplementary original data (excluding fluid-feeding consumers), the mean isotopic shift for C was +0.5±0.13‰ rather than 0.0‰, as commonly assumed. The shift for C was higher for consumers analyzed as muscle (+1.3±0.30‰) than for consumers analyzed whole (+0.3±0.14‰). Among consumers analyzed whole, the trophic shift for C was lower for consumers acidified prior to analysis (−0.2±0.21‰) than for unacidified samples (+0.5±0.17‰). For N, trophic shift was lower for consumers raised on invertebrate diets (+1.4±0.21‰) than for consumers raised on other high-protein diets (+3.3±0.26‰) and was intermediate for consumers raised on plant and algal diets (+2.2±0.30‰). The trophic shift for S differed between high-protein (+2.0±0.65‰) and low-protein diets (-0.5±0.56‰). Thus, methods of analysis and dietary differences can affect trophic shift for consumers; the utility of stable isotope methods can be improved if this information is incorporated into studies of trophic relationships. Although few studies of stable isotope ratios have considered variation in the trophic shift, such variation is important because small errors in estimates of trophic shift can result in large errors in estimates of the contribution of sources to consumers or in estimates of trophic position.     

High resolution genetic and physical mapping of the I-3 region of tomato chromosome 7 reveals almost continuous microsynteny with grape chromosome 12 but interspersed microsynteny with duplications on Arabidopsis chromosomes 1, 2 and 3

The tomato I-3 gene introgressed from the Lycopersicon pennellii accession LA716 confers resistance to race 3 of the fusarium wilt pathogen Fusarium oxysporum f. sp. lycopersici. We have improved the high-resolution map of the I-3 region of tomato chromosome 7 with the development and mapping of 31 new PCR-based markers. Recombinants recovered from L. esculentum cv. M82 × IL7-2 F2 and (IL7-2 × IL7-4) × M82 TC1F2 mapping populations, together with recombinants recovered from a previous M82 × IL7-3 F2 mapping population, were used to position these markers. A significantly higher recombination frequency was observed in the (IL7-2 × IL7-4) × M82 TC1F2 mapping population based on a reconstituted L. pennellii chromosome 7 compared to the other two mapping populations based on smaller segments of L. pennellii chromosome 7. A BAC contig consisting of L. esculentum cv. Heinz 1706 BACs covering the I-3 region has also been established. The new high-resolution map places the I-3 gene within a 0.38 cM interval between the molecular markers RGA332 and bP23/gPT with an estimated physical size of 50–60 kb. The I-3 region was found to display almost continuous microsynteny with grape chromosome 12 but interspersed microsynteny with Arabidopsis thaliana chromosomes 1, 2 and 3. An S-receptor-like kinase gene family present in the I-3 region of tomato chromosome 7 was found to be present in the microsyntenous region of grape chromosome 12 but was absent altogether from the A. thaliana genome. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.