High-level expression, purification, and characterization of recombinant wheat xylanase inhibitor TAXI-I secreted by the yeast Pichia pastoris

Triticum aestivum xylanase inhibitor I (TAXI-I) is a wheat protein that inhibits microbial xylanases belonging to glycoside hydrolase family 11. In the present study, recombinant TAXI-I (rTAXI-I) was successfully produced by the methylotrophic yeast Pichia pastoris at high expression levels (approximately 75 mg/L). The rTAXI-I protein was purified from the P. pastoris culture medium using cation exchange and gel filtration chromatographic steps. rTAXI-I has an iso-electric point of at least 9.3 and a mass spectrometry molecular mass of 42,013 Da indicative of one N-linked glycosylation. The recombinant protein fold was confirmed by circular dichroism spectroscopy. Xylanase inhibition by rTAXI-I was optimal at 20-30 degrees C and at pH 5.0. rTAXI-I still showed xylanase inhibition activity at 30 degrees C after a 40 min pre-incubation step at temperatures between 4 and 70 degrees C and after 2 h pre-incubation at room temperature at a pH ranging from 3.0 to 12.0, respectively. All tested glycoside hydrolase family 11 xylanases were inhibited by rTAXI-I whereas those belonging to family 10 were not. Specific inhibition activities against family 11 Aspergillus niger and Bacillus subtilis xylanases were 3570 and 2940IU/mg protein, respectively. The obtained biochemical characteristics of rTAXI-I produced by P. pastoris (no proteolytical cleft) were similar to those of natural TAXI-I (mixture of proteolytically processed and non-processed forms) and non-glycosylated rTAXI-I expressed in Escherichia coli. The present results show that xylanase inhibition activity of TAXI-I is only affected to a limited degree by its glycosylation or proteolytic processing.     

1,25-Dihydroxyvitamin D3 inhibits ultraviolet B-induced apoptosis,Jun kinase activation,and interleukin-6 production in primary human keratinocytes

We investigated the capacity of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] to protect human keratinocytes against the hazardous effects of ultraviolet B (UVB)-irradiation, recognized as the most important etiological factor in the development of skin cancer. Cytoprotective effects of 1,25(OH)(2)D(3) on UVB-irradiated keratinocytes were seen morphologically and quantified using a colorimetric survival assay. Moreover, 1,25(OH)(2)D(3) suppressed UVB-induced apoptotic cell death. An ELISA, detecting DNA-fragmentation, demonstrated that pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM for 24 h reduced UVB-stimulated apoptosis by 55-70%. This suppression required pharmacological concentrations 1,25(OH)(2)D(3) and a preincubation period of several hours. In addition, 1,25(OH)(2)D(3) also inhibited mitochondrial cytochrome c release (90%), a hallmark event of UVB-induced apoptosis. Furthermore, we demonstrated that 1,25(OH)(2)D(3) reduced two important mediators of the UV-response, namely, c-Jun-NH(2)-terminal kinase (JNK) activation and interleukin-6 (IL-6) production. As shown by Western blotting, pretreatment of keratinocytes with 1,25(OH)(2)D(3) 1 microM diminished UVB-stimulated JNK activation with more than 30%. 1,25(OH)(2)D(3) treatment (1 microM) reduced UVB-induced IL-6 mRNA expression and secretion with 75-90%. Taken together, these findings suggest the existence of a photoprotective effect of active vitamin D(3) and create new perspectives for the pharmacological use of active vitamin D compounds in the prevention of UVB-induced skin damage and carcinogenesis.     

Effect of diets rich in N-3 polyunsatured fatty acids on muscle lipids and fatty acids in Belgian Blue double-muscled young bulls

The present study was aimed at investigating the effect of duration and time of feeding n-3 fatty acids on the fatty acid composition of intramuscular fat and adipose tissue of bulls at slaughter. Four groups of bulls were given during three periods different diets, mainly differing in the presence of linseed as the predominant n-3 fatty acid source in the concentrate either or not in combination with grass (silage) as the roughage. The results show that the fatty acid composition of the feed during the earlier periods of life of the animal were important and influenced the final intramuscular fatty acid composition. Feeding n-3 PUFA during the phases before the finishing diet increased the long chain n-3 PUFA (C20:5n-3, C22:5n-3 and C22:6n-3) compared to animals which were fed only a C 18:3n-3 rich concentrate in the finishing period. The cis-9,trans-11CLA content was increased by feeding linseed in the fattening period and was mainly deposited in the triacylglycerol fraction of the intramuscular fat.     

Gerotranscendence in the second half of life: first empirical investigation in Flanders

Tornstam regards gerotranscendence as the final stage in a natural process towards maturity and wisdom, that can be described as a shift in meta-perspective which accompanies the process of aging: from a materialistic and rational perspective to a more cosmic and transcendent one. The present study contributes to the development of a by Tornstam used gerotranscendence scale, and looks for theoretically expected relations between gerotranscendence and psychosocial variables such as life satisfaction, death attitude, and recently experienced life crises. The relation between gerotranscendence and dimensions of religion and faith will also be examined. Participants were 120 older adults, 58 men and 62 women, aged 45-92 years. The scores on the gerotranscendence scale were not congruent, neither with the hypothetical threefold apriori scales, nor with the factor structure Tornstam had found in his research. An exploratory factor analysis yielded three factors which were to a certain extent reconcilable with the gerotranscendence theory. The subscales are Transcendent connection, Active involvement and coherence, and Attachment to life and material goods. Older respondents and respondents who experienced one or more life crises, have higher scores on Transcendent connection. The experience of connection seems to be related to dimensions of religion and faith. The results warrant further research into a measurement of gerotranscendence and the relation with dimensions of religion and faith.     

Design of a synthetic adhesion protein by grafting RGD tailed cyclic peptides on bovine serum albumin

A synthetic adhesion protein was designed by chemical grafting of the RGD tailed cyclic peptide cyclo[-d-Val-Arg-Gly-Asp-Glu(-Ahx-Tyr-Cys-NH₂)-] on the carrier protein bovine serum albumin (BSA). The cyclic conformation of the RGD motif grafted on the protein mimics the conformation of the motif displayed in native adhesion proteins such as fibronectin. The adhesion of the cells on polystyrene coated with the conjugate BSA–peptide was similar or even better than the one obtained when the proadhesive protein fibronectin was coated on the plates. Results also indicated that covalent coupling of the peptide on BSA is not absolutely required, since simple adsorption of the peptide on the protein coated on plates was efficient for enhancing cell adhesion. These results show that polystyrene support can be reconditioned with conformationally constrained RGD peptides to enhance cell adhesion on solid supports. The same methodology can be adapted for the development of new biomaterials based on the recognition of specific peptides.     

Differential protein expression profile in the liver of pikeperch (Sander lucioperca) larvae fed with increasing levels of phospholipids

A comparative proteomic approach was used to assess the protein expression profile in the liver of 34 days old pikeperch larvae fed from day 10 post hatching, with three isoproteic and isolipidic formulated diets varying by their phospholipid (PL) contents (% dry diet weight): 1.4% (PL1), 4.7% (PL5) and 9.5% (PL9). Using 2D-DIGE minimal labelling of liver extracts, we were able to show 56 protein spots with a differential intensity (p < 0.05) depending on the dietary PL content. Among these spots, 11 proteins were unambiguously identified using nanoLC-MS/MS tandem mass spectrometry. In the PL9 larvae, our results indicate that the glycolytic pathway could be down-regulated due to the under-expression of the fructose biphosphate aldolase B and the phosphoglucomutase 1. Meanwhile, propionyl coenzyme A carboxylase (a gluconeogenic enzyme) was under-expressed. In addition, another gluconeogenic and lipogenic enzyme, pyruvate carboxylase, was identified in 3 different spots as being under-expressed in fish fed with the intermediate PL level (PL5). A high PL content increased the expression of sarcosine dehydrogenase, an enzyme involved in methionine metabolism, along with vinculin, a structural protein. Moreover, several stress proteins (glutathione S-transferase M, glucose regulated protein 75 and peroxiredoxin-1) were modulated in response to the dietary PL level and fatty acid composition. In the larvae fed with the lowest dietary PL content (PL1), over-expression of both GSTM and GRP75 might indicate a cellular stress in this experimental treatment, while the under-expression of Prx1 might indicate a lower defence against oxidative stress. In conclusion, this nutriproteomic approach showed significant modifications of protein expression in the liver of pikeperch larvae fed different PL contents, highlighting the importance of these nutrients and their influence on metabolism processes and on stress response.     

Folding properties of the hepatitis B core as a carrier protein for vaccination research

The hepatitis B core (HBc) protein has been used successfully in numerous experiments as a carrier for heterologous peptides. Folding and capsid formation of the chimeric proteins is not always achieved easily. In silico analyses were performed to provide further comprehension of the feasibility for predicting successful capsid formation. In contrast to previous work, we show that common in silico predictions do not ensure assembly into particles. We included new considerations regarding capsid formation of HBc fusion proteins. Not only the primary sequence and the length of the inserts seem important, also the rigidity, the distance between the N and the C-terminus and the presence of cysteines, which could form disulphide bonds, could influence proper capsid formation. Furthermore, new conformational insights were formulated when linkers were added to create extra flexibility of the chimeric particles. Different hypotheses were suggested to clarify the obtained results. To this extent, the addition of glycine-rich linkers could lower high rigidity of the insert, removal of the strain of the core protein or ease interaction between the HBc and the insert. Finally, we observed specific changes in capsid formation properties when longer linkers were used. These findings have not been reported before in this and other virus-like particle carriers. In this study, we also propose a new high-yield purification protocol for fusion proteins to be used in vaccination experiments with the carrier protein or in comparative studies of particulate or non-particulate HBc fusion proteins.     

Citrullinated vimentin as an important antigen in immune complexes from synovial fluid of rheumatoid arthritis patients with antibodies against citrullinated proteins

Introduction  

Rheumatoid arthritis (RA) is an inflammatory disease, which results in destruction of the joint. The presence of immune complexes (IC) in serum and synovial fluid of RA patients might contribute to this articular damage through different mechanisms, such as complement activation. Therefore, identification of the antigens from these IC is important to gain more insight into the pathogenesis of RA. Since RA patients have antibodies against citrullinated proteins (ACPA) in their serum and synovial fluid (SF) and since elevated levels of citrullinated proteins are detected in the joints of RA patients, citrullinated antigens are possibly present in IC from RA patients.