Purification of p47f,a Necrosis‐Inducing Protein Fraction from the Secretome of Phytophthora capsici

The oomycete Phytophthora capsici causes wilting disease in chilli pepper and another solanaceous plants, with important economic consequences. Although much investigation has been conducted about this pathogen, little is still known about which of its proteins are involved in the infection process. In this study, the bioassay‐guided fractionation of the secretome of P. capsici resulted in the purification of a phytotoxic protein fraction designated as p47f, capable of inducing wilting and necrosis on leaves of Capsicum chinense Jacq, and having a 47 kDa polypeptide with proteolytic activity as the major component. The isolated p47f fraction induced DNA degradation and decreased cell survival of C. chinense cell suspension culture. Sequencing of p47f indicated the presence of 15 proteins, which could be grouped into seven classes including a protease group, cell wall remodelling proteins and the transglutaminase elicitor M81D, among others. This is the first report of P. capsici secreting proteins that modulate cell responses mediated by ROS in the host.     

Production of alginate by Azotobacter vinelandii grown at two bioreactor scales under oxygen-limited conditions

The oxygen transfer rate (OTR) was evaluated as a scale-up criterion for alginate production in 3- and 14-L stirred fermentors. Batch cultures were performed at different agitation rates (200, 300, and 600 rpm) and airflow rates (0.25, 0.5, and 1 vvm), resulting in different maximum OTR levels (OTR_max). Although the two reactors had a similar OTR_max (19 mmol L^?1 h^?1) and produced the same alginate concentration (3.8 g L^?1), during the cell growth period the maximum molecular weight of the alginate was 1,250 kDa in the 3-L stirred fermentor and 590 kDa in 14-L stirred fermentor. The results showed for the first time the evolution of the molecular weight of alginate and OTR profiles for two different scales of stirred fermentors. There was a different maximum specific oxygen uptake rate between the two fermenters, reaching 8.3 mmol g^?1 h^?1 in 3-L bioreactor and 10.6 mmol g^?1 h^?1 in 14-L bioreactor, which could explain the different molecular weights observed. These findings open the possibility of using $ q_{{{\text{O}}_{ 2} }} $ instead of OTR_max as a scaling criterion to produce polymers with similar molecular weights during fermentation.     

Disulfide bond polymerization of a cyclic peptide derived from the surface loop 4 of class 1 OMP of Neisseria meningitidis

Two peptides derived from the surface loop 4 of class1 Outer Membrane Protein (OMP) of Neisseriameningitidis were synthesized on solid phase usingthe Boc/Bzl strategy: one containing the entire loop4 cyclized and the other representing the polymerizedcyclic loop 4. To test a more efficient cyclic peptidepresentation, in the present study astrategy was developed to obtain polymers of cyclic peptides. Inorder to obtain the polymeric cyclic peptide, twoprotecting groups for cysteine were used – Acm andMob. The Cys(Acm)-protected cyclic peptide wasobtained after removing the Mob group. Thepolymerization reaction was carried out bysimultaneous deprotection/oxidation of S-Acmwith iodine. Analysis of the polymeric cyclic peptidein Tris-tricine-SDS-PAGE showed different bandswith molecular weights higher than expected for thecorresponding monomeric cyclic peptide. Both peptideswere used in immunization of four different mouse strains.The antisera raised against the peptides wereevaluated by ELISA and Western blotting vs. OMPpreparation of N. meningitidis. The titersraised against the polymerized cyclic peptide werehigher than the ones raised against the cyclicpeptide. The antisera elicited did not showbactericidal activity. Nevertheless, the antiseraelicited against the polymeric cyclic peptide in theCBA/J mouse strain showed opsonic activity. Theantibodies raised against the polymeric cyclic peptidewere successfully used as probes in Western blottingexperiments to verify the display of loop 4 peptide onthe surface of filamentous phage M13.     

ICOS Gene Polymorphisms (IVS1 + 173 T/C and c. 1624 C/T) in Primary Sjögren’s Syndrome Patients: Analysis of ICOS Expression

Background: Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease, which affects exocrine glands. T cell activation is a trigger mechanism in the immune response. Hyperreactivity of T cells and antibody production are features in pSS. ICOS can be critical in the pathogenesis of pSS. Methods: A total of 134 pSS patients and 134 control subjects (CS) were included. Genotyping was performed by PCR-RFLP. ICOS mRNA expression was quantified by real-time PCR, and CD4+ ICOS+ T cells were determined by flow cytometry. Results: The ICOS IVS1 + 173 T>C polymorphisms were not associated with susceptibility to pSS (p = 0.393, CI = 0.503–1.311). However, the c.1624 C>T polymorphism was associated with a reduction in the risk of development of pSS (p = 0.015, CI = 0.294–0.884). An increase in ICOS mRNA expression in patients was observed (3.7-fold). Furthermore, pSS patients showed an increase in membranal-ICOS expression (mICOS). High expression of mICOS (MFI) was associated with lymphocytic infiltration. Conclusions: The IVS1 + 173 polymorphism is not a genetic marker for the development of pSS, while c.1624 T allele was associated with a low risk. However, elevated mICOS expression in pSS patients with high lymphocytic infiltration was found. ICOS may have an important role in the immunopathogenesis of pSS and should be analyzed in T cell subsets in pSS patients as a possible disease marker.