Plasmid DNA vaccination

Plasmid DNA vaccination against tuberculosis is a very powerful and easy method for the induction of strong humoral responses, CD4+ mediated secretion of Th1 cytokines and CD8+ mediated CTL activity in mice. Tuberculosis DNA vaccines have not been assessed so far in humans, and clinical trials with DNA in general have been somewhat disappointing. However, numerous studies have reported on the potent priming capacity of plasmid DNA for Th1 and CD8+ mediated immune responses, which can be boosted subsequently by recombinant protein or recombinant pox-viruses. With respect to tuberculosis, prime/boost regimens with Mycobacterium bovis BCG vaccine are particularly promising and warrant further analysis.     

The influence of tapioca on the growth,the activity of glucoamylase and pigment production of <Emphasis Type="Italic">Monascus purpureus</Emphasis> UKSW 40 in soybean-soaking wastewater

The present study evaluates the usefulness of tapioca starch as additional carbon source for the growth of Monascus purpureus in soybean-soaking wastewater (SSW). The result revealed that M. purpureus grown on 2.0% (w/v) tapioca starch in SSW produced significantly (P < 0.05) higher amounts of biomass and production of the pigments (OD₄₀₀ and OD₅₀₀) when compared to those grown on glucose-or maltose-containing media. However, the glucoamylase activity of M. purpureus grown on the tapioca-SSW medium was not significantly increased when compared to those from the glucose-containing medium.     

Selection of Heterodera glycines chorismate mutase-1 alleles on nematode-resistant soybean

The soybean cyst nematode Heterodera glycines is the most destructive pathogen of soybean in the Unites States. Diversity in the parasitic ability of the nematode allows it to reproduce on nematode-resistant soybean. H. glycines chorismate mutase-1 (Hg-CM-1) is a nematode enzyme with the potential to suppress host plant defense compounds; therefore, it has the potential to enhance the parasitic ability of nematodes expressing the gene. Hg-cm-1 is a member of a gene family where two alleles, Hg-cm-1A and Hg-cm-1B, have been identified. Analysis of the Hg-cm-1 gene copy number revealed that there are multiple copies of Hg-cm-1 alleles in the H. glycines genome. H. glycines inbred lines were crossed to ultimately generate three F2 populations of second-stage juveniles (J2s) segregating for Hg-cm-1A and Hg-cm-1B. Segregation of Hg-cm-1A and 1B approximated a 1:2:1 ratio, which suggested that Hg-cm-1 is organized in a cluster of genes that segregate roughly as a single locus. The F2 H. glycines J2 populations were used to infect nematode-resistant (Hartwig, PI88788, and PI90763) and susceptible (Lee 74) soybean plants. H. glycines grown on Hartwig, Lee 74, and PI90763 showed allelic frequencies similar to Hg-cm-1A/B, but nematodes grown on PI88788 contained predominately Hg-cm-1A allele as a result of a statistically significant drop of Hg-cm-1B in the population. This result suggests that specific Hg-cm-1 alleles, or a closely linked gene, may aid H. glycines in adapting to particular soybean hosts.     

UreG, a chaperone in the urease assembly process, is an intrinsically unstructured GTPase that specifically binds Zn2+

Bacillus pasteurii UreG, a chaperone involved in the urease active site assembly, was overexpressed in Escherichia coli BL21(DE3) and purified to homogeneity. The identity of the recombinant protein was confirmed by SDS-PAGE, protein sequencing, and mass spectrometry. A combination of size exclusion chromatography and multiangle and dynamic laser light scattering established that BpUreG is present in solution as a dimer. Analysis of circular dichroism spectra indicated that the protein contains large portions of helices (15%) and strands (29%), whereas NMR spectroscopy indicated the presence of conformational fluxionality of the protein backbone in solution. BpUreG catalyzes the hydrolysis of GTP with a kcat=0.04 min(-1), confirming a role for this class of proteins in coupling energy requirements and nickel incorporation into the urease active site. BpUreG binds two Zn2+ ions per dimer, with a KD=42 +/- 3 microm, and has a 10-fold lower affinity for Ni2+. A structural model for BpUreG was calculated by using threading algorithms. The protein, in the fully folded state, features the typical structural architecture of GTPases, with an open beta-barrel surrounded by alpha-helices and a P-loop at the N terminus. The protein dynamic behavior observed in solution is critically discussed relative to the structural model, using algorithms for disorder predictions. The results suggest that UreG proteins belong to the class of intrinsically unstructured proteins that need the interaction with cofactors or other protein partners to perform their function. It is also proposed that metal ions such as Zn2+ could have important structural roles in the urease activation process.     

Diagonal reverse-phase chromatography applications in peptide-centric proteomics: ahead of catalogue-omics?

Diagonal electrophoresis/chromatography was described 40 years ago and was used to isolate specific sets of peptides from simple peptide mixtures such as protease digests of purified proteins. Recently, we have adapted the core technology of diagonal chromatography so that the technique can be used in so-called gel-free, peptide-centric proteome studies. Here we review the different procedures we have developed over the past few years, sorting of methionyl, cysteinyl, amino terminal, and phosphorylated peptides. We illustrate the power of the technique, termed COFRADIC (combined fractional diagonal chromatography), in the case of a peptide-centric analysis of a sputum sol phase sample of a patient suffering from chronic obstructive pulmonary disease (COPD). We were able to identify an unexpectedly high number of intracellular proteins next to known biomarkers.     

Probing the specificity of the subclass B3 FEZ-1 metallo-beta-lactamase by site-directed mutagenesis

The subclass B3 FEZ-1 beta-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the beta-lactam bond in penicillins, cephalosporins, and carbapenems. FEZ-1 has been extensively studied using kinetic, computational modeling and x-ray crystallography. In an effort to probe residues potentially involved in substrate binding and zinc binding, five site-directed mutants of FEZ-1 (H121A, Y156A, S221A, N225A, and Y228A) were prepared and characterized using metal analyses and steady state kinetics. The activity of H121A is dependent on zinc ion concentration. The H121A monozinc form is less active than the dizinc form, which exhibits an activity similar to that of the wild type enzyme. Tyr156 is not essential for binding and hydrolysis of the substrate. Substitution of residues Ser221 and Asn225 modifies the substrate profile by selectively decreasing the activity against carbapenems. The Y228A mutant is inhibited by the product formed upon hydrolysis of cephalosporins. A covalent bond between the side chain of Cys200 and the hydrolyzed cephalosporins leads to the formation of an inactive and stable complex.     

Identification of new antigenic peptide presented by HLA-Cw7 and encoded by several MAGE genes using dendritic cells transduced with lentiviruses

Antigens encoded by MAGE genes are of particular interest for cancer immunotherapy because they are tumor specific and shared by tumors of different histological types. Several clinical trials are in progress with MAGE peptides, proteins, recombinant poxviruses, and dendritic cells (DC) pulsed with peptides or proteins. The use of gene-modified DC would offer the major advantage of a long-lasting expression of the transgene and a large array of antigenic peptides that fit into the different HLA molecules of the patient. In this study, we tested the ability of gene-modified DC to prime rare Ag-specific T cells, and we identified a new antigenic peptide of clinical interest. CD8(+) T lymphocytes from an individual without cancer were stimulated with monocyte-derived DC, which were infected with a second-generation lentiviral vector encoding MAGE-3. A CTL clone was isolated that recognized peptide EGDCAPEEK presented by HLA-Cw7 molecules, which are expressed by >40% of Caucasians. Interestingly, this new tumor-specific antigenic peptide corresponds to position 212-220 of MAGE-2, -3, -6, and -12. HLA-Cw7 tumor cell lines expressing one of these MAGE genes were lysed by the CTL, indicating that the peptide is efficiently processed in tumor cells and can therefore be used as target for antitumoral vaccination. The risk of tumor escape due to appearance of Ag-loss variants should be reduced by the fact that the peptide is encoded by several MAGE genes.