Association of physical activity with all-cause mortality and incident and prevalent cardiovascular disease among patients with type 1 diabetes: the EURODIAB Prospective Complications Study.

AIMS/HYPOTHESIS: The aim of this study was to examine the association of physical activity (PA) with all-cause mortality and incident and prevalent cardiovascular disease (CVD) among patients with type 1 diabetes. METHODS: The EURODIAB Prospective Complications Study is a cohort including 3,250 male and female patients with type 1 diabetes (mean age 32.7 ± 10.2 years) from 16 European countries, of whom 1,880 participated in follow-up examinations. In analysis 1 (longitudinal), the association of baseline PA (based on the reported number of hours per week spent in mild, moderate and vigorous PA) with all-cause mortality and incident CVD was examined by performing survival analysis. In analysis 2 (cross-sectional), we focused on the association between PA at follow-up (data on sports, walking distance and regular bicycling) and prevalent CVD by performing logistic regression analysis. Adjustments were made for age, sex, BMI, smoking, consumption of alcohol, consumption of certain nutrients and diabetic complications. RESULTS: Analysis 1 (longitudinal): participation in moderate or vigorous PA once a week or more was borderline inversely associated with all-cause mortality (men and women combined) (HR 0.66, 95% CI 0.42, 1.03) and incident CVD (women only) (HR 0.66, 95% CI 0.40, 1.08). No association was found in men. Analysis 2 (cross-sectional): total PA (indexed by sports, walking, bicycling) and distance walked were inversely associated with prevalent CVD (OR(totalPA) 0.66, 95% CI 0.45, 0.97; and OR(walking) 0.61, 95% CI 0.42, 0.89). CONCLUSIONS/INTERPRETATION: PA showed a borderline inverse association with both all-cause mortality (both sexes) and incident CVD (women only) in patients with type 1 diabetes. Since this is an under-researched clinical population, future longitudinal studies with objective PA measurements are needed to expand on these results.

Harmonisation of food categorisation systems for dietary exposure assessments among European children.

Within the European project called EXPOCHI (Individual Food Consumption Data and Exposure Assessment Studies for Children), 14 different European individual food consumption databases of children were used to conduct harmonised dietary exposure assessments for lead, chromium, selenium and food colours. For this, two food categorisation systems were developed to classify the food consumption data in such a way that these could be linked to occurrence data of the considered compounds. One system served for the exposure calculations of lead, chromium and selenium. The second system was developed for the exposure assessment of food colours. The food categories defined for the lead, chromium and selenium exposure calculations were used as a basis for the food colour categorisation, with adaptations to optimise the linkage with the food colour occurrence data. With this work, an initial impetus was given to make user-friendly food categorisation systems for contaminants and food colours applicable on a pan-European level. However, a set of difficulties were encountered in creating a common food categorisation system for 14 individual food consumption databases that differ in the type and number of foods coded and in level of detail provided about the consumed foods. The work done and the problems encountered in this project can be of interest for future projects in which food consumption data will be collected on a pan-European level and used for common exposure assessments.

Prefoldin recognition motifs in the nonhomologous proteins of the actin and tubulin families.

Nascent actin and tubulin molecules undergo a series of complex interactions with chaperones and are thereby guided to their native conformation. These cytoskeletal proteins have the initial part of the pathway in common: both interact with prefoldin and with the cytosolic chaperonin containing tailless complex polypeptide 1. Little is understood with regard to how these chaperones and, in particular, prefoldin recognize the non-native forms of these target proteins. Using mutagenesis, we provide evidence that beta-actin and alpha-tubulin each have two prefoldin interaction sites. The most amino-terminally located site of both proteins shows striking sequence similarity, although these proteins are nonhomologous. Very similar motifs are present in beta- and gamma-tubulin and in the newly identified prefoldin target protein actin-related protein 1. Actin-related proteins 2 and 3 have related motifs, but these have altered charge properties. The latter two proteins do not bind prefoldin, although we identify them here as target proteins for the cytosolic chaperonin. Actin fragments containing the two prefoldin interaction regions compete efficiently with actin for prefoldin binding. In addition, they also compete with tubulins, suggesting that these target proteins contact similar prefoldin subunits.

The cytosolic class II chaperonin CCT recognizes delineated hydrophobic sequences in its target proteins.

The nonhomologous proteins actin and alpha- and beta-tubulin need the assistance of the cytosolic chaperonin containing TCP-1 (CCT) to reach their correct native state, and their folding requires a transient binary complex formation with CCT. We show that separate or combined deletion of three delineated hydrophobic sequences in actin disturbs the interaction with CCT. These sites are situated between residues 125-179, 244-285, and 340-375. Also, alpha- and beta-tubulin contain at least one recognition region, and intriguingly, it has a similar distribution of hydrophobic residues as region 244-285 in actin. Internal deletion of the sites in actin favor a model for cooperative binding of target proteins to CCT. Peptide mimetics, representing the binding regions, inhibit target polypeptide binding to CCT, suggesting that actin and tubulin contact similar CCT subunits. In addition, we show that actin recognition by class II chaperonins is different from that by class I.

Gene silencing results in instability of antibody production in transgenic plants.

The stability of antibody and Fab expression was assessed in five different homozygous transgenic Arabidopsis lines. Each of these lines showed silencing of the transgenes that encode the antibody polypeptides, leading to instability of antibody production. However, each line had a different and specific instability profile. The characteristic variation in the level of antibody accumulation in each line as a function of developmental stage indicated that the T-DNA integration pattern played a role in triggering silencing, and also that the history and the integration position of simple transgene loci can influence the susceptibility to epigenetic silencing. In different lines with low antibody accumulation levels, methylation was found either in the promoter alone, in both the promoter and the transcribed region, in the transcribed region only, or in the transcribed region and downstream sequences. In conclusion, our data suggest that epigenetic effects result in different transgene expression profiles in each of the five Arabidopsis lines analyzed.

Accumulation pattern of IgG antibodies and Fab fragments in transgenic Arabidopsis thaliana plants.

For the further optimization of antibody expression in plants, it is essential to determine the final accumulation sites of plant-made antibodies. Previously, we have shown that, upon secretion, IgG antibodies and Fab fragments can be detected in the intercellular spaces of leaf mesophyll cells of transgenic Arabidopsis thaliana plants. However, immunofluorescence microscopy showed that this is probably not their final accumulation site. In leaves, IgG and Fab fragments accumulate also at the interior side of the epidermal cell layers and in xylem vessels. These accumulation sites correspond with the leaf regions where water of the transpiration stream is entering a space impermeable to the proteins or where water is evaporating. In roots, plant-made Fab fragments accumulate in intercellular spaces of cortex cells, in the cytoplasm of pericycle and, to a lesser extent, endodermis cells, and in cells of the vascular cylinder. In other words, antibody accumulation occurs at the sites where water passes on its radial pathway towards and within the vascular bundle. Taken together, our results suggest that, upon secretion of plant-made antibodies or Fab fragments, a large proportion of these proteins are transported in the apoplast of A. thaliana, possibly by the water flow in the transpiration stream.

Use of phage display for isolation and characterization of single-chain variable fragments against dihydroflavonol 4-reductase from Petunia hybrida.

To isolate specific single-chain variable (scFv) fragments against dihydroflavonol 4-reductase (DFR) from Petunia hybrida the phage display technology was used. DFR was overproduced in Escherichia coli, purified and used for immunization. From DFR-immunized mice, a phage display library was made starting from spleen mRNA using an optimized set of primers for V(H) and V(L) amplification. Several rounds of panning against recombinant DFR yielded five different scFv fragments, confirmed by subsequent DNA sequencing. They all specifically bound to recombinant DFR in ELISA and DFR in flower extracts on Western blot. These results show that phage display is a promising technology in plant molecular biology to obtain specific recombinant antibodies not only for ELISA and Western blot but also for in vivo applications in the long run.

T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs.

Nicotiana protoplasts and Arabidopsis leaf discs or roots were co-cultivated with two Agrobacterium strains each carrying a different T-DNA. Co-transformed plants were selected and the integration of the different T-DNAs was analysed at the genetic and genomic level. Genetic analysis showed that the T-DNAs derived from different bacteria were frequently integrated at the same locus, independent of the plant species or transformation method used. Southern analysis revealed that 12 out of 27 Arabidopsis transformants contained the co-transferred T-DNAs linked to each other in all possible configurations but with a preference for those with at least one right border involved in linkage. Overall, our data support the hypothesis that ligation of separate T-DNAs is a dominant mechanism in formation of the frequently observed repeats of identical T-DNAs. We propose a scheme which could explain the formation of T-DNA repeats and the preferential involvement of right borders in T-DNA linkages.

Bacterial and plant-produced scFv proteins have similar antigen-binding properties.

A gene encoding a single-chain variable (scFv) antibody fragment was expressed as a cytoplasmic and endoplasmic reticulum-targeted protein in transgenic tobacco plants. In both cases, the scFv accumulated up to 0.01% of total soluble protein (TSP). The same scFv fragment was also produced in the periplasm of Escherichia coli. Measurement of the affinity by ELISA indicates that the affinity of the bacterially made scFv is about 80-fold lower than that of the parental Fab fragment. The results suggest that the affinity of the plant-produced scFv fragments is reduced to a similar extent, implying that all the plant-produced scFv fragments are antigen binding.

Assembly of an antibody and its derived antibody fragment in Nicotiana and Arabidopsis.

The yield and assembly of an IgG1 antibody and its derived F(ab) fragment were compared in Nicotiana and Arabidopsis. The results obtained showed a lot of interclonal variability. For 45% of the primary transgenic calluses, antigen-binding entities represented less than 0.1% of the total soluble protein (TSP). Only two of the 103 analysed transformants contained more than 1% of antigen-binding protein, with 1.26% being the highest yield. Analogous amounts of complete antibody and F(ab) accumulated in primary callus tissue. Moreover, yields were in the same range for both species as far as primary callus tissue is concerned. However, the accumulation of the F(ab) fragment in leaf tissue of regenerated plants differed significantly between Nicotiana and Arabidopsis. The F(ab) fragment accumulated to only 0.044% of TSP in Nicotiana leaves but up to 1.3% in Arabidopsis leaves. Furthermore, both species showed differences in the assembly pattern of the complete antibody. Whereas Arabidopsis contained primarily fully assembled antibodies of 150 kDa, Nicotiana showed an abundance of fragments in the 50 kDa range.

[Lumbar hypermobility: where swimming becomes hydrotherapy]. / Lumbale hypermobiliteit: waar zwemmen hydrotherapie wordt.

In this paper the authors discuss the clinical problem of lumbar hypermobility. The therapeutical possibilities are resumed briefly. The philosophy of medical training therapy ("Heilgymnastik") is described. More extensive the extra-advantages of hydrotherapy (methodical back-stroke swimming) are searched for in a theoretical deductive way. The authors found that: 1. swimming is a low-impact sport so far as the articulations are concerned, 2. back-stroke is done mainly in a lumbar kyphosis, 3. swimming is also an excellent cardiopulmonary training, 4. when swimming the muscles of the shoulder girdle and pelvic girdle are trained in a nearly isokinetic way (power-endurance), 5. the short transverso-spinal muscles are indirectly trained in their tonic more than phasic stretch reflex (posture function), 6. the muscles of the trunk are trained in a nearly isometric way in the appropriate angles (erect position), 7. the position of the head in the water facilitates the abdominal muscles (tonic neck reflex), 8. the cool temperature of the water generates training-enhancing stress-responses, 9. endurance-training is ideal for the postural function of the lower back muscles (especially the deeper layers near the spine) which are anatomical and physiological suited for this purpose, 10. warming-up and cooling-down procedures prepare the neuromuscular, the cardiovascular and metabolic functions before the workout-session (a cold shower afterwards acts to tonicize the skin and muscles).