Population studies of the wild tomato species Solanum chilense reveal geographically structured major gene-mediated pathogen resistance

Natural plant populations encounter strong pathogen pressure and defence-associated genes are known to be under selection dependent on the pressure by the pathogens. Here, we use populations of the wild tomato Solanum chilense to investigate natural resistance against Cladosporium fulvum, a well-known ascomycete pathogen of domesticated tomatoes. Host populations used are from distinct geographical origins and share a defined evolutionary history. We show that distinct populations of S. chilense differ in resistance against the pathogen. Screening for major resistance gene-mediated pathogen recognition throughout the whole species showed clear geographical differences between populations and complete loss of pathogen recognition in the south of the species range. In addition, we observed high complexity in a homologues of Cladosporium resistance (Hcr) locus, underlying the recognition of C. fulvum, in central and northern populations. Our findings show that major gene-mediated recognition specificity is diverse in a natural plant-pathosystem. We place major gene resistance in a geographical context that also defined the evolutionary history of that species. Data suggest that the underlying loci are more complex than previously anticipated, with small-scale gene recombination being possibly responsible for maintaining balanced polymorphisms in the populations that experience pathogen pressure.     

Use of isoelectric focusing to define major histocompatibility complex class I polymorphism in goats

We have used biochemical methods to extend and improve serological class I typing using a panel of 77 Swiss goats of the Saanen breed, comprising dam-offspring combinations from six half-sib sire families and several unrelated animals. Of these animals class I molecules were precipitated from cell lysates with the mAb B1.1G6 and HC10. Immunoprecipitates were analysed by SDS-PAGE and 1D-IEF. There was a good agreement between class I serological types and IEF banding patterns. We have identified three new class I specificities and subdivided the Bel 7 specificity. IEF has enabled us to make planned immunizations to produce antisera to the new specificities. New evidence for the expression of a second class I locus product in the Be7 haplotype has been found.     

Calibration of EMG to force for knee muscles is applicable with submaximal voluntary contractions.

PURPOSE: In this study, the influence of using submaximal isokinetic contractions about the knee compared to maximal voluntary contractions as input to obtain the calibration of an EMG-force model for knee muscles is investigated. METHODS: Isokinetic knee flexion and extension contractions were performed by healthy subjects at five different velocities and at three contraction levels (100%, 75% and 50% of MVC). Joint angle, angular velocity, joint moment and surface EMG of five knee muscles were recorded. Individual calibration values were calculated according to [C.A.M. Doorenbosch, J. Harlaar, A clinically applicable EMG-force model to quantify active stabilization of the knee after a lesion of the anterior cruciate ligament, Clinical Biomechanics 18 (2003) 142-149] for each contraction level. RESULTS: First, the output of the model, calibrated with the 100% MVC was compared to the actually exerted net knee moment at the dynamometer. Normalized root mean square errors were calculated [A.L. Hof, C.A.N. Pronk, J.A. van Best, Comparison between EMG to force processing and kinetic analysis for the calf muscle moment in walking and stepping, Journal of Biomechanics 20 (1987) 167-187] to compare the estimated moments with the actually exerted moments. Mean RMSD errors ranged from 0.06 to 0.21 for extension and from 0.12 to 0.29 for flexion at the 100% trials. Subsequently, the calibration results of the 50% and 75% MVC calibration procedures were used. A standard signal, representing a random EMG level was used as input in the EMG force model, to compare the three models. Paired samples t-tests between the 100% MVC and the 75% MVC and 50% MVC, respectively, showed no significant differences (p>0.05). CONCLUSION: The application of submaximal contractions of larger than 50% MVC is suitable to calibrate a simple EMG to force model for knee extension and flexion. This means that in clinical practice, the EMG to force model can be applied by patients who cannot exert maximal force.     

Toll-like receptor 2 pathway drives streptococcal cell wall-induced joint inflammation: critical role of myeloid differentiation factor 88

The IL-1R/Toll-like receptor (TLR) superfamily of receptors has a key role in innate immunity and inflammation. In this study, we report that streptococcal cell wall (SCW)-induced joint inflammation is predominantly dependent on TLR-2 signaling, since TLR-2-deficient mice were unable to develop either joint swelling or inhibition of cartilage matrix synthesis. Myeloid differentiation factor 88 (MyD88) is a Toll/IL-1R domain containing adaptor molecule known to have a central role in both IL-1R/IL-18R and TLR signaling. Mice deficient for MyD88 did not develop SCW-induced arthritis; both joint swelling and disturbance of cartilage chondrocyte anabolic function was completely abolished. Local levels of proinflammatory cytokines and chemokines in synovial tissue washouts were strongly reduced in MyD88-deficient mice. Histology confirmed the pivotal role of MyD88 in acute joint inflammation. TLR-2-deficient mice still allow influx of inflammatory cells into the joint cavity, although the number of cells was markedly reduced. No influx of inflammatory cells was seen in joints of MyD88-deficient mice. In addition, cartilage matrix proteoglycan loss was completely absent in MyD88 knockout mice. These findings clearly demonstrated that MyD88 is a key component in SCW-induced joint inflammation. Since agonists of the Toll-like pathway are abundantly involved in both septic and rheumatoid arthritis, targeting of MyD88 may be a novel therapy in inflammatory joint diseases.     

Analysis of protein-carbohydrate interaction at the lower size limit of the protein part (15-mer peptide) by NMR spectroscopy,electrospray ionization mass spectrometry,and molecular modeling

Structural analysis of minimally sized lectins will offer insights into fundamentals of intermolecular recognition and potential for biomedical applications. We thus moved significantly beyond the natural limit of lectin size to determine the structure of synthetic mini-lectins in solution, their carbohydrate selectivity and the impact of ligand binding on their conformational behavior. Using three disaccharide (Thomsen-Friedenreich antigen; Gal beta 1,3GalNAc alpha 1,R)-binding pentadecapeptides without internal disulfide bridges as role models, we successfully tested a combined strategy with different techniques of NMR spectroscopy, electrospray ionization mass spectrometry, and molecular modeling. In solution, the peptides invariably displayed flexibility with rather limited restrictions, shown by NMR experiments including nearly complete resonance assignments and molecular dynamics simulations. The occurrence of aromatic/nonpolar amino acids in the sequence did not lead to formation of a hydrophobic core known from microbial chitinase modules. Selectivity of disaccharide binding was independently observed by mass spectrometry and NMR analysis. Specific ligand interaction yielded characteristic NMR signal alterations but failed to reduce conformational flexibility significantly. We have thereby proven effectiveness of our approach to analyze even low-affinity interactions (not restricted to carbohydrates as ligands). It will be useful to evaluate the impact of rational manipulation of lead peptide sequences.     

Rapid duplex PCR assay for the detection of pathogenic Yersinia enterocolitica strains

For the detection of pathogenic Yersinia enterocolitica strains, a duplex PCR has been developed based on differences observed between the fingerprint profiles of pathogenic and non-pathogenic strains. The profiles were obtained by using a primer derived from the Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences. From the sequence of one pathogen-specific amplified fragment, a discriminative primer has been designed bridging the sequence of the highly conserved core region and 3' end of the ERIC element. In combination with three other primers, all located within the detected open reading frame that resembled the sequence of the bipA gene, this primer was applied in a duplex PCR assay to simultaneously detect Y. enterocolitica and to discriminate between pathogenic and non-pathogenic strains. The same primer combinations were used in an on line rapid cycling real-time PCR assay. The used SYBR Green I format allowed for the easy translation of the PCR conditions and confirmation of the resulting amplicons. The time of analysis was reduced to approximately 60 min.     

TLR-3 is Present in Human Adipocytes,but Its Signalling is Not Required for Obesity-Induced Inflammation in Adipose Tissue In Vivo

Innate immunity plays a pivotal role in obesity-induced low-grade inflammation originating from adipose tissue. Key receptors of the innate immune system including Toll-like receptors-2 and -4 (TLRs) are triggered by nutrient excess to promote inflammation. The role of other TLRs in this process is largely unknown. In addition to double-stranded viral mRNA, TLR-3 can also recognize mRNA from dying endogenous cells, a process that is frequently observed within obese adipose tissue. Here, we identified profound expression of TLR-3 in adipocytes and investigated its role during diet-induced obesity. Human adipose tissue biopsies (n=80) and an adipocyte cell-line were used to study TLR-3 expression and function. TLR-3-/- and WT animals were exposed to a high-fat diet (HFD) for 16 weeks to induce obesity. Expression of TLR-3 was significantly higher in human adipocytes compared to the non-adipocyte cells part of the adipose tissue. In vitro, TLR-3 expression was induced during differentiation of adipocytes and stimulation of the receptor led to elevated expression of pro-inflammatory cytokines. In vivo, TLR-3 deficiency did not significantly influence HFD-induced obesity, insulin sensitivity or inflammation. In humans, TLR-3 expression in adipose tissue did not correlate with BMI or insulin sensitivity (HOMA-IR). Together, our results demonstrate that TLR-3 is highly expressed in adipocytes and functionally active. However, TLR-3 appears to play a redundant role in obesity-induced inflammation and insulin resistance.     

Oxaloacetate hydrolase, the C-C bond lyase of oxalate secreting fungi

Oxalate secretion by fungi is known to be associated with fungal pathogenesis. In addition, oxalate toxicity is a concern for the commercial application of fungi in the food and drug industries. Although oxalate is generated through several different biochemical pathways, oxaloacetate acetylhydrolase (OAH)-catalyzed hydrolytic cleavage of oxaloacetate appears to be an especially important route. Below, we report the cloning of the Botrytis cinerea oahA gene and the demonstration that the disruption of this gene results in the loss of oxalate formation. In addition, through complementation we have shown that the intact B. cinerea oahA gene restores oxalate production in an Aspergillus niger mutant strain, lacking a functional oahA gene. These observations clearly indicate that oxalate production in A. niger and B. cinerea is solely dependent on the hydrolytic cleavage of oxaloacetate catalyzed by OAH. In addition, the B. cinera oahA gene was overexpressed in Escherichia coli and the purified OAH was used to define catalytic efficiency, substrate specificity, and metal ion activation. These results are reported along with the discovery of the mechanism-based, tight binding OAH inhibitor 3,3-difluorooxaloacetate (K(i) = 68 nM). Finally, we propose that cellular uptake of this inhibitor could reduce oxalate production.     

Longitudinal Study of Performance on the Ruff Figural Fluency Test in Persons Aged 35 Years or Older

The Ruff Figural Fluency Test (RFFT) is a cognitive test to measure executive function. Longitudinal studies have shown that repeated testing improves performance on the RFFT. Such a practice effect may hinder the interpretation of test results in a clinical setting. Therefore, we investigated the longitudinal performance on the RFFT in persons aged 35–82 years. Performance on the RFFT was measured three times over an average follow-up period of six years in 2,515 participants of the Prevention of REnal and Vascular ENd-stage Disease (PREVEND) study in Groningen, the Netherlands: 53% men; mean age (SD), 53 (10) years. The effect of consecutive measurements on performance on the RFFT was investigated with linear multilevel regression models that also included age, gender, educational level and the interaction term consecutive measurement number x age as independent variables. It was found that the mean (SD) number of unique designs on the RFFT increased from 73 (26) at the first measurement to 79 (27) at the second measurement and to 83 (26) at the third measurement (p<0.001). However, the increase per consecutive measurement number was negatively associated with age and decreased with 0.23 per one-year increment of age (p<0.001). The increase per consecutive measurement number was not dependent on educational level. Similar results were found for the median (IQR) number of perseverative errors which showed a small but statistically significant increase with repeating testing: 7 (3–13) at the first measurement, 7 (4–14) at the second measurement and 8 (4–15) at the third measurement (p_trend = 0.002). In conclusion, the performance on the RFFT improved by repeating the test over an average follow-up period of three to six years. This practice effect was the largest in young adults and not dependent on educational level.